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Full text of "Analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide"

ANALGESIC AND IMMUNOMODULATORY EFFECTS OF 
CODEINE AND CODEINE 6-GLUCURONIDE 



By 
VINAYAK JAYA SRINIVASAN 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1996 



i.~' ■■.... 






>\ ? ^ ; i -- / ACKNOWLEDGMENTS 

I would like to take this opportunity to offer my heartfelt appreciation to Dr. 
Ian Tebbett. His continous guidance and encouragement was the driving force 
for this research project. I would like to express my sincere thanks to the other 
members of my committee-Dr. Donna Wielbo, Dr. Kenneth Sloan, Dr. Hartmut 
Derendorf and Dr. Roger Bertholf-for all their advice and suggestions throughout 
the project. 

I want to thank Dr. James Simpkins, Dr. Janet Karlix and Dr. Guenther 
Hochhaus for giving me an opportunity to work in their labs in order to generate 
some of my data. I also would like to thank Jim Ketcham for his invaluable help 
in putting all my posters, papers and this dissertation together. 

I would also like to convey my thanks to Shawn Toffolo and Becky 
Frieburger for their help in my work. I also deeply appreciate the support of all 
the graduate students and the other faculty members and secretaries in the 
department. 

I want to express my deepest gratitute to my brother, my relatives and all 
my friends who have stayed by me through the thick and thin. Finally, I want to 
dedicate this work to my parents because I did it for them. 



TABLE OF CONTENTS 

pagg 
ACKNOWLEDGMENTS ii 

ABSTRACT vi 

CHAPTERS 

1. BACKGROUND AND SIGNIFICANCE 1 

1.1 Opioids and Pain 2 

1.1.1 Pain Transnnission 3 

1.1.2. Pain Perception 4 

1.2 Codeine 4 

1.2.1 Administration and Dosage 5 

1.2.2 Pharmacological Actions 6 

1.2.3 Toxicity 7 

1.2.4 Therapeutic Uses 7 

1.2.5 Drug Dependence and Tolerance 8 

1.2.6 Analytical Techniques 9 

1.2.7 Pharmacokinetics in Man 10 

1.2.7.1 Absorption 11 

1.2.7.2 Distribution 11 

1.2.7.3 Metabolism 12 

1.2.7.4 Elimination 14 

1.2.8 Pharmacokinetics in Rats 16 

1.2.8.1 Absorption 16 

1.2.8.2 Distribution 17 

1.2.8.3 Metabolism 18 

1.2.8.4 Elimination 19 

1.3 Drug Glucuronidation 20 

1.3.1 Overview 20 

1.3.2 Direct Pharmacological Activity 21 

1.4 Evaluation of Analgesia in Small Animals 26 

1.4.1 Introduction 26 



III 



1.4.2 Time Course of Analgesic Effect 27 

1.5 Genetic Polymorphism 28 

1.6 Immunomodulation 29 

1.6.1 Opioid Receptors 30 

1.6.2 Effects on Lymphocytes 31 

1.6.3 Effects on Myleoid Cells 32 

1 .6.4 Effects on Natural Killer Cells 34 

1.6.5 Mechanism of Action 34 

1.7 Receptor Binding 37 

1.8 Hypotheses 39 

1.9 Specific Objectives 39 

2. METHODS 41 

2.1 Specific Objective #1 : Analytical Method 41 

2.1.1 Materials 41 

2.1.2 Extraction Procedure 41 

2.1.2.1 Human urine 41 

2.1.2.2 Rat plasma 42 

2.1.2.3 Rat brain 42 

2.1.3 Chromatographic Conditions 43 

2.1.3.1 HPLC system 1 43 

2.1.3.2 HPLC system 2 44 

2.2 Specific Objective #2: Synthesis of Codeine 6-glucuronide 45 

2.2.1 Reaction Step I 45 

2.2.1.1 Dry benzene 47 

2.2.1.2 Fresh silver carbonate 47 

2.2.2 Reaction Step II 48 

2.2.3 Reaction Step III 48 

2.3 Specific Objective #3: Analgesic Activities of Codeine and 

Codeine 6-glucuronide 48 

2.3.1 Intracerebroventhcular Route Studies 49 

2.3.1.1 Surgery 50 

2.3.1.2 Tail flick method 50 

2.3.2 Subcutaneous Route Studies 51 

2.3.3 Intravenous Route Studies 51 

2.3.4 Statistics 52 

2.4 Specific Objective #4: Immune Studies with Human 

T Lymphocytes 52 

2.4.1 Method 53 

2.4.2 Statistics 55 

2.5 Specific Objective #5: Receptor Binding Studies 55 



IV 



2.5.1 Materials 55 

2.5.2 Method 56 

2.6 Specific Objective #6: Plasma and Brain Concentrations 57 

3. RESULTS 59 

3.1 HPLC Development 59 

3.1.1 Extraction Recoveries 61 

3.1.2 Range / Linearity of Standard Curve 61 

3.1.3 Specificity 62 

3.1.4 Sensitivity / Limit of Detection and Quantitation 63 

3.1.5 Precision and Accuracy 63 

3.1.6 Stability 65 

3.2 Synthesis 65 

3.3 Analgesia Studies 66 

3.3.1 Intracerebroventricular Route 66 

3.3.2 Subcutaneous Route 70 

3.3.3 Intravenous Route 83 

3.4 Immune Studies 105 

3.5 Receptor Binding Studies 111 

3.6 Plasma and Brain Concentrations 116 

4. DISCUSSION 121 

REFERENCES .....:...... 132 

BIOGRAPHICAL SKETCH 149 



-, f 



^■t 






Abstract of Dissertation Presented to the Graduate School 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



ANALGESIC AND IMMUNOMODULATORY EFFECTS OF 
CODEINE AND CODEINE 6-GLUCURONIDE 

By 

VINAYAK JAYA SRINIVASAN 

May 1996 

Chairman: Dr. Ian Ronald Tebbett 
Major Department: Pharmaceutics 

The interactions between opioid analgesics and the human immune 
system can have important clinical consequences. A better understanding of 
these interactions is needed due to the widespread use and abuse of opiates. In 
recent years, an increased knowledge and awareness in this area has generated 
a considerable surge in research. Narcotics are predominantly used to alleviate 
pain and discomfort in patients with trauma or undergoing major surgery. 
However, they are also known to cause impairment of the immune system. 
Subsequently, this could lead to patients becoming predisposed to infectious 
diseases as a result of the immunosuppressive effects of narcotics. 

An HPLC system was successfully developed for the analysis of codeine 
and its metabolites in various biological samples, that is, plasma, urine and brain 
tissue. Codeine 6-glucuronide and an intermediate compound were synthesized 
using a modification of the Koenigs-Knorr reaction. The synthetic procedure was 



VI 



efficient and reproducible. Analgesia studies with the tail flick method showed 
that codeine 6-glucuronide and the intermediate exhibited a higher analgesic 
activity compared to codeine when administered intracerebroventricularly. 
However, both compounds were not as active as codeine when administered by 
subcutaneous and intravenous routes. Immunomodulatory studies showed that 
the glucuronide metabolites of codeine and morphine were less 
immunosuppressive compared to their parent compounds, especially at 
physiologically relevant concentrations. Receptor binding profiles of codeine 6- 
glucuronide and the intermediate were similar to codeine, indicating that they 
possessed activity towards the |i-opioid receptors. 

The overall goal of the project was to correlate the analgesic and 
immunomodulatory effects of codeine and codeine 6-glucuronide. This would 
result in a better understanding of the significance of high levels of codeine 6- 
glucuronide present in the plasma and urine in man after codeine administration. 
Further, this may lead to the development of glucuronide analogs for the 
management and treatment of pain in immunocompromised patients. 



VII 



CHAPTER 1 
BACKGROUND AND SIGNIFICANCE 



Pain is an unpleasant sensation that can disturb the comfort, thought, 
sleep, and normal daily activity of a person. Pain signals are considered to be 
part of a protective mechanism designed to indicate the presence of a potentially 
dangerous condition. Thus, it is considered to be symptomatic of an underlying 
condition that requires attention and treatment. Pain is the net effect of complex 
interactions of ascending and descending neurosystems which include 
biochemical, physiological, psychological, and neurocortical processes. Also, 
since pain is a very subjective experience, only the patient can describe its 
intensity. This subjectivity makes it difficult to assess the activity of analgesics in 
humans. 

Analgesics are defined as drugs that can relieve pain without causing loss 
of consciousness. The most potent analgesics are referred to as narcotics and 
act directly on the central nervous system. Narcotics as a group include the 
opioids, which are considered to be the most effective analgesics available. The 
opioid family, whose name derives from opium, includes agents such as 
morphine, codeine, meperidine and methadone. While opioid is a general term 
for any drug, natural or synthetic, that has actions similar to morphine, the term 
opiate is more specific and applies only to compounds present in opium such as 
morphine and codeine. Apart from acting as analgesics, opioids produce a 
variety of pharmacological actions on various tissues in the body. 



1.1 Opioids and Pain 

Opioids represent the main class of drugs in the clinical management of 
mild to moderate pain in various cases of medical illness, and relieve pain 
primarily through direct actions on receptors in the central nervous system. 
Opioid analgesics include natural alkaloids from opium (morphine, codeine), 
synthetic surrogates (methadone, meperidine) and endogenous peptides 
(enkephalins, p-endorphins). , . 

Opioids act at receptor sites both within and outside the central nervous 
system. Binding studies with various drugs and ligands in the brain and other 
tissues suggest the presence of a multitude of opioid receptors. The three 
important receptor types are designated as mu {\x), kappa (k) and delta (5). The 
effects mediated by the |i receptors include supraspinal analgesia, respiratory 
depression and euphoria. The k receptors mediate analgesia at the level of the 
spinal cord, along with sedation and miosis. The 5 receptors are also thought to 
be involved in analgesia, both at the spinal and supraspinal sites. However, their 
role in this regard remains controversial (Jaffe and Martin, 1985). 

The body produces three families of peptides that are capable of 
interacting with opioid receptors-enkephalins, (3-endorphins and dynorphins. 
These endogenous opioids have a high affinity for the ^, k and 5 receptors, 
respectively. Ttiey are present throughout the body and serve as hormones and 
neurotransmitters. It is thought that morphine and other opioid analgesics mimic 
the actions of these endogenous ligands by binding to the opioid receptors. 
These interactions are presumed to give rise to the observed pharmacological 
effects. 



1.1.1 Pain T ransmission 

Pain generally begins with a noxious stimulus that injures or destroys 
tissues. Endogenous chemical substances such as histamine, bradykinins, 
prostaglandins, and others are then released from the damaged tissues and 
nerve terminals. The released chemicals bind to "pain receptors" or nociceptors 
present along the afferent nerve fibers, depolarizing the nerve membranes and 
initiating an action potential. This causes the generation of a pain impulse which 
is then transmitted via the afferent fibers to the spinal cord as shown in the figure 
below (Figure 1-1). When the pain signals arrive at the spinal cord, they are in 
turn relayed to the higher centers of the brain-thalamus and cortex. 



Descending 
Modulation 



MIDBRAIN 



Nucleus 
Raptie Magnus 



MEDULLA 



Sp4r«o- Thalamic 
Tract 



Pen-AQu«^uctal Oray 



Lateral Reticular 
Formalion 



Medial Reticular 
Formation 




From 

Primary Afferent 

Pain Fibres 



Figure 1-1 : The pain modulating system (adapted from Puntillo, 1988). 






»>■■■ *h 



1.1.2 Pain Perception 

Although some responses are reflexive in nature (e.g., knee jerk), the 
perception and appraisal of pain usually occurs in the higher centers of the brain. 
These systems are known to be responsible for attention, mood, motivation and 
arousal. Thus, pain is perceived in the thalamic and forebrain levels and 
evaluated in the cortex (Puntillo, 1988). 

The perception and reaction to pain varies with each individual. It is now 
evident that a host of biochemical substances, including neurotransmitters and 
endogenous opioids, can modulate pain by either facilitating or inhibiting the 
transmission of pain impulses at various levels of the nervous system. There is 
evidence that the pain suppression system is mediated in part by endogenous 
opioids along the descending pathway, which relays processed information in 
response to the pain stimulus (Figure 1-1). The administration of exogenous 
opioids, like morphine and codeine, is thought to enhance this pain suppression 
system. However, the exact relationship between the analgesic effect of opioids 
and the role of pain modulators is yet to be clearly established. 

1.2 Codeine 

Codeine (Figure 1-2) is a naturally occurring alkaloid in which the phenolic 
hydroxy! group of morphine is replaced by a methoxy group. It was isolated in 
1832 by Robiquet from the opium plant, papaver somniferum. Barbier in 1834 
was the first to report its analgesic activity in humans (Baselt and Cravey, 1989). 



H3C0 




HO 



Figure 1-2 : The structure of codeine (adapted from Muhtadi and Hassan, 
1981). 

Codeine is a white crystalline powder which, when made anhydrous, 
melts at 154-156 °C. The phosphate salt is more soluble than the sulfate salt and 
hence it is used more commonly. The free base is sparingly soluble in water, but 
freely soluble in alcohol. Codeine is a monoacidic, weak base with a pKa value 
of 8.2 (Baselt and Cravey, 1989). It exhibits a characteristic UV absorbance peak 
in water at 284.8 nm (Grasselli and Ritchey, 1975). The anhydrous base has a 
nominal molecular weight of 300. 

1.2.1 Administration and Dosage 

Codeine is usually given orally as a phosphate or sulfate salt for the relief 
of cough and mild to moderate pain. The phosphate salt may also be given 
parentally for the relief of pain by intramuscular or subcutaneous injection. 

As an analgesic, the usual oral dose is 30 to 60 mg every four or six 
hours, as needed for the relief of pain. For treatment of cough, the usual adult 
dose is 10 to 20 mg every four to six hours, not to exceed 120 mg. As with other 



opiate agonists, the smallest effective dose must be given in order to minimize 
the development of tolerance and physical dependence. 

1.2.2 Pharmacological Actions 

Codeine, like morphine, acts by blocking excitatory synaptic transmission 
in the central nervous system and relieves pain and anxiety primarily by raising 
the pain threshold. It exerts a combination of depressing and stimulating effects 
on the central nervous system and various peripheral organs. Important CNS 
effects include analgesia, euphoria, sedation and respiratory depression. 
Supression of the cough reflex is a well-recognized action of opioids, particularly 
codeine. Miosis (constriction of pupils) is another pharmacological action seen 
with virtually all opioid agonists. Codeine can also cause activation of the brain 
stem chemoreceptor trigger zone to produce nausea and vomiting. 

Peripheral effects of codeine include increasing the tone and decreasing 
the rhythmic contractions of different types of smooth muscles. In the 
gastrointestinal tract this produces constipation, which may be troublesome in 
ordinary analgesic therapy, but useful in the treatment of diarrhea (Jaffe and 
Martin, 1985). Urethral and biliary tract spasms are usually increased by 
codeine, but the analgesia produced may outweigh these undesirable effects. It 
can, however, be life threatening in cases of asthma when combined with 
repiratory depression. 

Other effects of codeine include central vasomotor capacity depression 
and dilatation of some vessels, including the coronary arteries. On the whole, 
these circulatory effects are small and probably result from a combination of 



central actions and peripheral histamine release. Some anticholinergic activity 
may be present, but is probably not critical (Way and Adier, 1962). 

1.2.3 Toxicity " .'^ :■ 

Codeine shares the toxic potentials of the opiate agonists. The most 
common side effects observed after the administration of therapeutic doses of 
codeine include dizziness, sedation, nausea, vomiting, sweating and a feeling of 
light-headedness. Other adverse effects that can be seen include euphoria, 
dysphoria, weakness, headache, insomnia, anorexia, gastrointestinal distress, 
bradycardia and even urinary retention. 

Toxic effects of opioid overdose produce a classic triad of signs : coma, 
respiratory depression and constricted pupils. Breathing becomes shallow and 
irregular and may slow to as low as 2-4 breaths per minute. A severe overdose 
of codeine can cause respiratory depression, cyanosis, extreme somnolence 
which can progress to a coma, and severe hypotension with bradycardia. This 
could lead to apnea, circulatory collapse, cardiac failure and finally death. 
Codeine toxicity can be treated successfully with an intravenous administration 
of the narcotic antagonist naloxone (Cutting, 1972 ; Jaffe and Martin, 1985 ; 
McEvoy, 1990). 

1 .2.4 Therapeutic Uses 

Codeine is a mild analgesic indicated for symptomatic relief of moderate 
pain. It is considered to be 1/10 to 1/6 as potent as morphine as an analgesic. It 
is also used as an antitussive, alone or in combination with other antitussives or 



8 



expectorants, in the symptomatic relief of non-productive cough (Cutting, 1972 ; 
Jaffe and Martin, 1985 ; McEvoy, 1990). 

1.2.5 Drug Dependence and Tolerance 

The major limitation of opioids is that they characteristically produce "drug 
habituation" or "addiction". Drug dependence is marked by tolerance-the gradual 
deveopment of resistance to the effects of the drug after repeated administration. 
Tolerance is manifested by a decline in the effectiveness of a drug, requiring a 
gradual increase in the dosage in order to maintain the initial effect. 

Psychic dependence is a clinical term to indicate habituation. It is defined 
as compulsive use of a drug by an individual who feels euphoric and a sense of 
well-being from its chronic use. This kind of dependence is seen to a lesser or 
greater extent with numerous agents like caffeine, nicotine, salicylates and 
bromides as well as narcotic analgesics. 

Physical dependence, on the other hand, deals with the biochemical and 
physiological adaptation of tissues to a new chemical environment after repeated 
use of a drug. The drug becomes necessary for normal tissue function and its 
withdrawal causes an abnormal cellular response referred to as "abstinence 
syndrome". This situation is usually characterized by effects opposite to those of 
the pharmacological effects of the drug. 

The phenomenon of physical dependence can actually be visualized as 
being due to the prolonged occupation of the receptor sites within the cells of the 
central nervous system by opioid analgesics. This receptor-drug interaction leads 
to adaptive changes in the latent cellular excitability. These changes then 
manifest themselves during drug abstinence as symptoms of withdrawal. The 



intensity of the withdrawal syndrome is proportional to the amount and duration 
of drug administration. 

1.2.6 Analytical Techniques 

Until recently there was little pharmacokinetic data described in the 
literature regarding low doses of codeine. This was mainly due to the lack of 
analytical techniques of sufficient sensitivity and specificity. Earlier studies relied 
on colorimetric assays which were not very sensitive (Woods, Muehlenbeck and 
Mellett, 1956). Johannesson and Woods (1964), Yeh and Woods (1969, 1970) 
used high doses of radiolabeled codeine and were able to measure codeine and 
biotransformed morphine in rat plasma. 

As codeine undergoes extensive metabolism, forming active metabolites, 
there is a need to develop an assay to precisely determine the extent of 
formation of each metabolite and quantify its potential contribution to the overall 
analgesia and/or toxicity associated with codeine. The older analytical methods 
to determine the levels of codeine and some of its metabolites included 
radioimmunoassay (Findlay et al., 1977 ; Gintzler et a!., 1976), gas 
chromatography (Jain et al., 1977 ; Kogan and Chedchel, 1976) and gas 
chromatography-mass spectroscopy (Ebbighausen et al., 1973 ; Cone et al. 
1983) techniques. 

Although radioimmunoassays offer the sensitivity required for the 
detection of these compounds, differentiation between very similar species like 
morphine 6-glucuronide and morphine 3-glucuronide cannot be achieved. The 
ability to identify and quantify the above metabolites is important, since morphine 
6-glucuronide is known to be pharmacologically active. Gas chromatography and 



10 



mass spectroscopy can offer both sensitivity and specificity required for 
examination of codeine and morphine, but the techniques involve time- 
consuming derivatization steps and are not suitable for the glucuronides. Many 
researchers have turned their attention to the development of rapid, sensitive 
and specific HPLC methods for the detection of opiates. 

Numerous HPLC-based methods have been reported with ultraviolet 
(Persson et a!., 1989), fluorescence (Chen et al., 1989 ; Tsina et al., 1982) and 
electrochemical (Harris et al., 1988 ; Svensson 1986 ; Verway-van Wissen et al., 
1991 ; Besner et al., 1989 and Bedford and White, 1985) detection systems. 
HPLC with fluorescence detection requires the conversion of some compounds 
to fluorescent products before analysis. Electrochemical detection does not allow 
simultaneous detection of all the compounds, due to differing redox potentials. 
HPLC with ultraviolet detection would therefore seem to be the method of choice 
for developing an assay for all of the compounds of interest. 

A potential problem with using a reversed phase HPLC system is that the 
polar glucuronides elute very close to the solvent front and are prone to being 
hidden by co-eluting endogenous substances (Chari et al., 1991). An alternate 
method has been described for morphine and its metabolites using a normal 
phase system (Wielbo et al., 1993). 

1.2.7 Pharm acokinetics in Man 

There is an abundance of literature describing the pharmacokinetics of 
codeine in man (Quiding et al., 1986 ; Guay et al., 1987 ; Chen et al., 1991 ; Yue 
et al., 1989 a , 1990 a, b ; Shah and Mason, 1990 a ; Way and Adier, 1962 ; 
Findlay et al., 1977, 1978, 1986 ; Hull et al., 1982 ; Rogers et al., 1982 ; Persson 



11 



et al., 1992 ; Vree and Verway-van Wissen, 1992). Despite this extensive 
documentation, the relevance of some of the active metabolites of codeine is not 
clear. The extent of formation of individual metabolites and their potential 
contributions to the analgesic efficacy seen after codeine administration need to 
be assessed in detail. 

1.2.7.1 Absorption 

Codeine is well absorbed following oral and intramuscular administration 
in man. Its bioavailability after oral administration was found to be 50-60% 
(Rogers et al., 1982). At a dose of 60 mg, a peak plasma concentration (Cmax) 
of around 100-200 ng/ml was seen within one to two hours after administration 
(Mohammed et al., 1993). Chen et al. (1991) observed that the mean tmax (tifne 
at which the peak plasma concentration is observed) for codeine occurred about 
1 hour after oral administration in the case of both single and chronic dosing. 
However, the mean peak plasma concentration after chronic dosing was 
significantly higher than after single dosing. This was also observed by Quiding 
et al. (1986) and can in part explain why some subjects experience a greater 
analgesic effect after chronic dosing compared to single dosing. 

1.2.7.2 Distribution 

After the drug is absorbed into the blood, it is distributed to tissues in the 
body. However, only the free drug concentration can equilibrate with these 
tissues. The main interaction in blood is between the plasma proteins and the 
drug molecules and this is usually a reversible physical process. Thus, the 
binding of drugs to plasma proteins is a dynamic process. Codeine has been 
shown to be bound to plasma proteins to an extent of 25-30% (Findlay et a!., 
1977). Baselt and Cravey (1989) reported a volume of distribution of about 



12 



3.5 I/kg for codeine, indicating an extensive distribution in the various tissues of 
the body. 

1 ■2.7.3 Metabolism 

Codeine is primarily metabolized in the liver, the major site being the 
microsomes in the endoplasmic reticulum. Lesser, but significant sites include 
the central nervous system, kidney, lung and placenta. Metabolism is 
predominantly via conjugation with glucuronic acid at the 6-position (Yue et al., 
1989 a, b, 1990 a, b ; Chen et al., 1991 ; Vree and Venway-van Wissen, 1992). 
Other metabolic pathways include 0-demethylation to morphine and N- 
demethylation to norcodeine (Sindrup et al., 1990). These primary metabolites of 
codeine are further metabolized to their glucuronides as outlined in Figure 1-3. 

The hepatic biotransformation of codeine to morphine has led many to 
believe that that codeine may exert its analgesic effect through partial conversion 
to morphine (Adier and Latham, 1950 ; Findlay et al., 1978 ; Yue et al., 1990 a). 
This assumption is supported by the low affinity of codeine to the [i opiate 
receptor and by the marked in vivo analgesic efficacy of morphine, morphine-6- 
glucuronide and normorphine (Lasagna and Kornfeld, 1958 ; Osborne et al., 
1988). However, studies by Quiding et al. (1986) and Shah and Mason (1990 a) 
have questioned the possible role of morphine in codeine analgesia because of 
the very low concentrations of morphine seen after both single and repeated 
doses of codeine. 

Chen et al. (1991) described codeine 6-glucuronide pharmacokinetics in 
detail after single and chronic oral codeine administration. There was no 
difference in the plasma tmsx (1.28 versus 1.13 h) and Cmax (1 43 versus 1.38 
(ig/ml) for single and chronic dosing, respectively. The mean AUCs for codeine 



13 



and codeine 6-glucuronide at steady state were not significantly different for 
either single or chronic dosing. The average ratio of the AUCc6G • AUCcod was 

Codeine-6-giucuronide 



Glucuronidation 
NCH3 




CH3O ' o 
0-demethyIation / Codeine 

_NCH3 



N-demethylation 

NH 



Glucuronidation 





OH . 



M3G, M6G Morphine 

-«. N-demethylation 




CH30 \ ■ 

Norcodeine 

O-demethylation 



Normorphine 

Figure 1-3 : The various metabolic pathways of codeine. 

about 15 for both single and chronic dosing regimens. Vree and Verway-van 
Wissen (1992) reported similar values for the various pharmacokinetic 
parameters of codeine 6-glucuronide except in the case of t-1/2 and AUC ratio. 






14 



While they reported ti/2 values for codeine and codeine 6-glucuronide as 1.5 
and 2.8 hours respectively, Chen's group found the t-1/2 values for both 
compounds to be similar, about 3 hours. Vree's group reported a AUCcsG • 
AUCcod '"3^'° °^ "10 compared with 15 by Chen's group. 

Quiding et al. (1986) reported plasma concentrations of morphine both 
after single and multiple doses of 60 mg of codeine to be about 2-3% of that of 
codeine. Shah and Mason (1990 a) found that Morphine AUG values ranged 
from 2-5% after a 60 mg oral dose of codeine. However, Vree and Verway-van 
Wissen (1992) reported that no free morphine could be detected in the plasma of 
human volunteers who took 30 mg of codeine orally. Any morphine formed was 
immediately glucuronidated at the 3- and 6- positions to form the corresponding 
glucuronides. Both glucuronides were detected in the plasma with the morphine 
3-glucuronide concentrations being higher than those of morphine 6-glucuronide. 
Very small amounts of normorphine, norcodeine and its glucuronide conjugate, 
norcodeine 6-glucuronide, were also detected in the plasma. 

1.2.7.4 Elimination 

Codeine and its metabolites are excreted almost exclusively by the 
kidneys. A very small fraction is eliminated as free codeine (about 5%). The 
major portion of the administered dose appearing in the urine consists of 
biotransformed products. Urinary recoveries of codeine and its metabolites 
indicate that codeine 6-glucuronide is the major metabolite formed from codeine. 
There are also trace amounts of morphine and its glucuronides along with 
normorphine and its glucuronide conjugate (Chen et al., 1991). The excretion of 
codeine and its metabolites in 24 hour urine as a% of the dose of administered 
codeine is summarized in Table 1-1. Adier et al. (1955) observed that, after a 



- - r • ' 

f^-V ..... . t 



15 



single dose of codeine, urinary excretion was almost complete in 24 hours, 
although trace amounts of codeine and morphine stayed in the body for several 
days before being completely eliminated. A small percentage of the dose (0.02- 
0.17%), consisting mostly of free codeine and some metabolites, was also 
detected in the feces. 



Codeine / Metabolites 


% Excreted in 24 Hour Urine 


Codeine 


8-16 


Morphine 


0.5-1 


Codeine 6-glucuronide 


48-69 


Morphine 6-glucuronide 


0.5-2 


Morphine 3-glucuronide 


5-8 


Norcodeine 


2-10 



Table 1-1 : The urinary excretion data in humans after a 60 mg oral dose 
of codeine, (from Yue et al., 1990 a). 



Codeine has an elimination half-life of about 2-3 hours (Findlay et a!., 
1977, 1978 ; Quiding et al., 1986 ; Yue et al., 1990 a, b). Chen et al. (1991) 
reported that elimination half-lives for both codeine (3.2 versus 2.9 h) and 
codeine 6-glucuronide (3.2 vs 3.3 h) after both single and chronic dosing, 
respectively, were not significantly different. The renal clearance of codeine is 
between 67 and 265 ml/min. The creatinine clearance values of codeine in 
healthy volunteers has been reported to be in the range of 90 to 132 ml/min., 
indicating that in addition to glomerular filtration, codeine can undergo active 
secretion into the lumen of the proximal tubules (Chen et al., 1991). 



16 



1.2.8 Pharmacokinetics in Rats 

The physiological disposition of codeine in various experimental animals 
after relatively high doses has been extensively studied. Studies done in male 
rats have shown that about half the dose (55%) of codeine undergoes O- 
demethylation (Yeh and Woods, 1969). Morphine formed by the metabolic 
conversion of codeine has been found in the plasma, urine, bile and feces 
(Johannesson and Shou, 1963 ; Johannesson and Woods, 1964 ; Yoshimura et 
al., 1970). Morphine has also been determined to be present in the brain of rats 
following large doses of codeine (Dahlstrom and Paalzow, 1976). 

Traditionally, the major routes of administration in rats have been 
subcutaneous (s.c.) and intraperitoneal (i.p.) injections. Numerous 
pharmacological experiments with rats to determine analgesic activity of codeine, 
morphine (Johannesson and Shou, 1963 ; Yeh and Woods, 1969 , 1970 ; Oguri 
et al., 1990) and the 3- and 6-glucuronide metabolites (Yoshimura et al., 1973 ; 
Shimomura et al., 1971 ; Abbott and Palmour, 1988 ; Sullivan et al., 1989 ; Gong 
et al., 1991 ; Paul et al., 1989 ; Pasternak et al., 1987 ; Smith et al., 1990), have 
utilized both s.c. and i.p. routes of administration. There are a few reports in the 
literature regarding intravenous administration of codeine and morphine in rats 
(Dahlstrom and Paalzow, 1976 ; Shah and Mason, 1991 ; Bhargava and Villar, 
1992 ; Thurston et al., 1993). Shah and Mason (1990 b) also administered 
codeine orally in rats as well as by i.v. injection and compared the two routes of 
administration. 
1.2.8.1 Absorption 

Shah and Mason (1990 b) described codeine pharmacokinetics after an 
oral dose of 5 mg/kg in rats. A solution of codeine phosphate was made by 



17 



dissolving it in 2-4 ml of physiological saline. The drug was then carefully 
delivered via gastric intubation to the fasting animals. Codeine was rapidly 
absorbed after the 5 mg/kg oral dose. The mean peak plasma concentrations 
were 101.3 ± 42.4 ng/ml around 6.4 + 4.5 min after dosing. After 4 hours, no 
codeine could be detected in the whole blood with the HPLC method used in the 
study. A large intersubject variation was observed in the absorption of codeine. 
This variation may be partly explained by factors such as gastric motility and 
intestinal transit time. 

The mean bioavailability was calculated from oral /\L/C; * Dj ^ / i.v. AUC^ 

* Doral 3S 0.08 ± 0.03, that is., only 8% of the ingested dose of codeine reaches 
the systemic circulation. Absorption of opiates in general is thought to occur by 
passive diffusion rather than by processes invoving energy expenditure 
(Christensen et al., 1984). Incomplete absorption was not considered to be a 
major factor as the amounts of free codeine found in the feces were negligible 
following oral administration. 

1. 2.8.2 Distribution 

Miller and Elliot (1955) conducted several distribution experiments in the 
rat. They showed that codeine was well distributed, and capable of leaving the 
the blood and concentrating in parenchymatous tissues such as liver, kidney, 
lung, adrenal glands and brain. This is reflected in the large volume of 
distribution of the terminal portion (Vd area ~ 5-1 I/kg), obtained from the 
concentration-time profile after intravenous codeine administration. 

Codeine given i.v. appears to fit a two compartment body model with a 
rapid distribution and a short terminal elimination phase. This indicates that 
codeine is rapidly metabolized and /or excreted. Elimination rate from the central 



18 



compartment (0.0569 min"'') is more than the terminal rate constant (P), due to 
distribution of codeine into the peripheral compartment. The mean ratio of 0.92 
for K-|2 / K21 (ratio of the inetrcompartmental rate constants) shows 
approximately an equivalent distribution of codeine between the central and 
peripheral compartments. Linear pharmacokinetics was exhibited by codeine at 
i.v. doses of 1-4 mg/kg (Shah and Mason, 1990 b). 
1.2.8.3 Metabolism 

There are a few studies in the literature that examine the metabolism of 
codeine in rats. These were done mostly in the 1960s and were conducted with 
radioactive codeine. Plasma concentrations after a 2 mg/kg s.c. injection of 
codeine showed the presence of free codeine, free morphine and conjugated 
morphine (in the ratio of 10 : 3 : 1). No conjugated codeine was seen either in 
the plasma or brain (Yeh and Woods, 1969). The conjugated morphine was later 
characterized as morphine 3-glucuronide. It was reaffirmed that morphine 6- 
glucuronide and codeine 6-glucuronide were not formed in rats (Yeh and Woods, 
1970). However, using thin layer chromatography, a Japanese group 
(Yoshimura, 1970) detected traces of codeine 6-glucuronide (0.2%) in the urine 
of rats, showing that a small amount of this metabolite can be formed in rats. 
This has been supported by Oguri et al. (1990) who used a specific HPLC 
method and found the urinary recovery of this compound to be about 1% (Table 
1-2). Recent findings (Oguri et al., 1990 ; Lawrence et al., 1992) have shown that 
morphine 6-glucuronide could not be detected in plasma or urine of rats after 
codeine administration. . ■ .. .. , . 






-f . 4; ' >> ^ 



19 



1. 2.8.4 Elimination 

In vivo codeine disposition studies using radiolabeled carbon-14 have 
shown that about 74% of the injected radioactivity is excreted as free codeine, 
free morphine and morphine conjugate via the pulmonary, biliary, intestinal and 
urinary routes in male rats (Yeh and Woods, 1969). Of this 74%, 20-40% is 
eliminated in the expired air as CO2 via the pulmonary pathway. 



Codeine / Metabolites 


% Excreted in 24 Hour Urine 


Codeine 


1 -2 


Morphine 


4-5 


Codeine 6-glucuronide 


0.1-0.3 1 


Morphine 6-glucuronide 


Not detected 


Morphine 3-glucuronide 


23-24 


1 Norcodeine 


Not detected 



Table 1-2 : Urinary excretion data as a% of a dose of codeine (from 
Ogurietal., 1990). 



A significant part of an i.v. dose of codeine has been shown to undergo 
enterohepatic recirculation to the extent of 10-30% (Walsh and Levine, 1975). It 
is known that codeine can decrease gastrointestinal tract motility, thereby 
increasing transit time of morphine glucuronide in the intestinal tract. This would 
then allow longer exposure to bacterial glucuronide hydrolysis which, in turn, 
would favor enterohepatic recycling. Yeh and Woods (1969), using radioactively- 
labeled tracers, reported the following amounts recovered from intact bile : free 
codeine (1.3%), free morphine (0.9%) and conjugated morphine (43.1%). 



20 



Oguri et al. (1990) saw a substantial interspecies difference in the 
metabolism of codeine (Table 1-3). This can influence considerably the nature 
and duration of the pharmacological and toxicological activities of codeine. The 
development of molecular aspects of gene evolution has been applied 
extensively to explain species differences seen in drug metabolism (Nebert and 
Gonzalez, 1987). 



Codeine/ IVIetabolites 


Mouse 

(n=25) 


Rat 

(n=4) 


Guinea Pig 

(n=4) 


Rabbit 

(n=3) 


Codeine 


6.8 + 0.7 


1.6 + 0.2 


1.6 + 0.2 


2.2 + 0.5 


Morphine 


0.8 + 0.2 


4.3 + 0.4 


0.2 + 0.1 


1.3 + 0.3 


Codeine 6-glucuronide 


1.6 + 0.2 


0.2 + 0.1 


39.8 + 3.9 


24.5 + 3.7 


Morphine 6-glucuronide 


nd 


nd 


0.7 + 0.1 


1.9 + 0.3 


Morphine 3-glucuronide 


7.6+ 1.0 


23.9 + 2.8 


1.6 + 0.2 


17.9 + 1.4 


Norcodeine 


9.0 + 0.8 


nd 


nd 


nd 



nd = not detected 



Table 1-3 : Urinary excretion data (% of dose) 24 hours after codeine 
administration in various species (from Oguri et al., 1990). 

1.3 Drug Glucuronidation 



1.3.1 Overview 

A major pathway for drug metabolism and excretion is the generation of 
water soluble glucuronide metabolites. Since many drugs exhibit structural 
features that allow conjugation without previous phase I reactions, 
glucuronidations are viewed as first line detoxification mechanisms. Most 



21 



glucuronides of drugs are considered to be inactive and rapidly eliminated. 
Therefore, glucuronide nnetabolites of drugs are often neglected in 
pharmacodynamic and pharmacokinetic studies and are not taken into account 
when evaluating drug effects. 

The pharmacological and toxicological relevance of glucuronidation was 
pioneered by Dutton and is summarized in a comprehensive review by Kroemer 
and Klotz (1992). The general reaction scheme which describes the conjugation 
of glucuronic acid to various drugs in the presense of glucuronosyl transferase 
enzymes is shown in Figure 1-4. This reaction mediates the formation of ether, 
ester, thiolic, N- and C- glucuronides. 

Uridine diphosphate glucuronosyltransferases (UGTs) are enzymes 
located in the endoplasmic reticulum and therefore form a part of the microsomal 
fraction. UGTs are generally 50 to 60 kD in size and span the entire membrane 
of the endoplasmic reticulum. There is a small C-terminal domain located in the 
cytoplasm with the active site directed toward the lumen of the endoplasmic 
reticulum. 

1.3.2 Direct Pharmacological Activitv 

The best documented example of glucuronide activity is that of morphine. 
Morphine is conjugated to give morphine 3-glucuronide and morphine 6- 
glucuronide in the liver (Wahlstrom et al., 1988 ; Coughtrie et al., 1989). 
Shimomura et al. (1971) observed that morphine 6-glucuronide had a direct 
analgesic effect in the hot plate test. Subsequently, the same group 
demonstrated that both morphine 6-glucuronide and morphine 3-glucuronide can 
penetrate the blood brain barrier (Yoshimura et al., 1973). Using the tail flick 



22 




"° I <\0 - (P) ®- OH2C s. /O. 



GA 



OH OH 



UOP 



UOP gfucuronosyl 
transferase 



COOH 




+ UDP 



Figure 1-4 : Glucuronidation of a substrate (H-S) by reaction with 
uridine diphosphate-glucuronic acid (UDP-GA) in the presence of UDP- 
glucuronosyl transferase enzymes (from Kroemer and Klotz, 1992). 



latency tests in rats, they found that morphine 6-glucuronide was 20-fold more 
potent than morphine after direct microinjection into the periaquaductal gray area 
of the brain. Morphine 3-glucuronide in this experimental design did not produce 
any effect. 

The observations of Pasternak have been confirmed by a number of 
investigators (Paul et al., 1989), who performed detailed characterization of 
morphine 6-glucuronide. After peripheral administration to rats the analgesic 
effect was twice that of morphine itself. After intrathecal administration, morphine 
6-glucuronide was reported to be 650 times more potent than the parent 
compound. Smith et al. (1990) showed that morphine 3-glucuronide had no 
analgesic activity but could act as a potent antagonist of morphine and morphine 
6-glucuronide induced analgesia in rats. In this context Woolf (1981) reported 
that morphine 3-glucuronide was capable of inducing hyperalgesia in rats. 



23 



Recent investigations of force field and quatum mechanical characterization of 
morphine 3-glucuronide and morphine 6-glucuronide reveal an unexpectedly 
high degree of lipophllicity (Carrupt et al., 1991). 

The question which then arises is whether this detailed pharmacological 
evidence for the contribution of glucuronides to the net drug effects of morphine 
is matched by clinical observations. Joel et al. (1985) speculated that morphine 
6-glucuronide may contribute to the clinical efficacy of morphine. Hanks et al. 
(1987) tried to explain the potency of repeated oral doses of morphine as due to 
accumulation of morphine 6-glucuronide. Direct clinical evidence for the 
analgesic action of morphine 6-glucuronide was obtained by Osborne et al. 
(1988), who injected morphine 6-glucuronide, 1.0 mg/kg to 5 patients and 0.5 
mg/kg to 1 patient. Five patients reported total pain relief within 30 minutes and 
the analgesia lasted for 1-7 hours. Pain and pain relief were monitored by visual 
analog scales (Figure 1-5). : 



Pharmacokinetic Parameters 


Patient A 

(Normal Renal 
Function) 


Patient B 11 

(Impaired Renal 
Function) 


Clearance (I/kg) ** 


89 


26 II 


Volume of Distribution (1) 


14.7 


16.4 


Elimination Half-life (h) ** 


1.9 


7.4 


1 Area Under the Curve (nmol r'h) ** 


370 


1319 



indicates that the parameters were significantly different. 



Table 1-4 : Pharmacokinetic parameters of two patients after a 1 mg/kg 
intravenous dose of morphine 6-glucuronide (from Osborne et al., 1988). 



24 



1 00 T 



^ 80 

u 

E 

3 

g 60 

'x 

CO 

E 



o 



40 



20 




TIME (hours) 



Figure 1-5 : A visual analog scale used for monitoring the extent of pain 
relief (from Osborne et al., 1988). 



250 T 




4 6 

TIME (hours) 



Figure 1-6 : The pharmacokinetic profile of two patients after 1 mg/kg i.v. 
administration of morphine 6-glucuronide (from Osborne et al., 1988). 



25 



Pharmacokinetic indices for two patients are shown in Table 1-4. Patient 
A had normal renal function while patient B had chronic renal impairment. The 
elimination of morphine 6-glucuronide was closely related to renal function. No 
morphine or morphine 3-glucuronide levels were detected in the plasma at any 
time. Plasma morphine 6-glucuronide levels for the two patients are shown in 
Figure 1-6. 

Hannah et al. (1990) investigated the analgesic efficacy of intrathecal 
morphine 6-glucuronide in comparison with morphine in 3 patients with chronic 
cancer pain. The doses required for controlled analgesia were 393 + 227 mg/24 
h and and 227 ± 114 mg/24 h for morphine and morphine 6-glucuronide 
administration, respectively. 

Conjugation of drugs by glucuronosyl transferases plays an important role 
in the overall picture of drug disposition. The resulting glucuronides represent 
metabolites that are not always inactive and may in fact contribute to drug action 
either directly (by producing analgesia as in the case of morphine 6-glucuronide) 
or indirectly (by release of the parent compound via hydrolysis as in 
enterohepatic recycling). Moreover, some glucuronic acid conjugates are not 
rapidly excreted. Their disposition can be modulated at different levels of 
distribution, metabolism and excretion, thereby modifying net drug action. 
Therefore, glucuronic acid conjugates should be taken into account when 
pharmacokinetic and pharmacodynamic characterization of drugs are 
determined. 



26 



1.4 Evaluation of Analgesia in Small Animals 

1.4.1 Introduction 

Eddy (1928, 1932) is credited as being the first to describe methods for 
determining analgesia in animal experiments by exerting a variable pressure on 
the distal part of the cat tail. Friend and Harris (1948) used a pair of forceps, 
whereby pressure could be exerted on the tail of the rat. Green et al. (1951) 
produced pain by exerting pressure on the tip of the tail using a syringe piston 
system. 

Macht and Macht (1940) were the first to describe a method in which 
electrical stimulation was used to produce pain in rats. They implanted two 
electrodes in the skin of the scrotum. The rats reacted with a squeak response 
when the voltage was increased over a certain threshold. Luckner and Magun 
(1951) implanted two electrodes in the upper part of the tail. Collins et al. (1964) 
implanted an electrode in the rectum and another in the upper part of the tail in 
rats. 

Eddy et al. (1950) placed mice in a cylindrical glass container, the base of 
which was a copper plate. This plate was maintained at a temperature of 50-55 
°C by a hot water bath placed below the copper plate. The mice responded to 
the heat by licking their forepaws and trying to jump out of the cylinder. This 
technique is called Eddy's hot plate method and is a good pain model for the rat 
and mouse. D'Amour and Smith (1941) irradiated the tail tip with heat from a light 
source of defined strength. This tail flick method is also widely used for 
determinating analgesia in small animals. 



27 



A modification of the tail flick method was described by Berglund and 
Simpklns (1988). It involves measurement of the withdrawal time of the tail when 
a beam of light was focused on it. This was done both before drug 
administration, and at regular intervals thereafter. The former measurement was 
called baseline latency and the latter test latency. The Instrument used was a 
model 33 tail flick analgesia meter (lite Inc., Landing, NJ, USA), and consisted of 
an incandescent light bulb with the beam intensity and sensitivity dial set at 75 
and 8, respectively. The time between presentation of a focused beam of light 
and removal of the tail was recorded as the latency period. However, in the 
absence of any response, a cut-off period of 40 seconds was used to prevent 
tissue damage. This was considered as the maximal suppression of pain. 

1.4.2 Time Course of Analgesic Effect 

D'Amour and Smith (1941) were the first to determine the analgesic effect 
of morphine and codeine administered by i.p. injection. They found the effect to 
be maximal at 30 minutes post-injection for both drugs. Ercoli and Lewis (1945) 
injected equianalgesic doses of morphine and codeine, both intraperitoneally 
and subcutaneously. They found that both drugs had their greatest effect 30-60 
minutes after administration, with the effect persisting for 60-120 minutes. 
Following higher doses, the degree of analgesia was found to be more complete. 

Miller and Elliott (1955) were the first researchers to make a serious 
attempt to look into changes in the amounts of codeine and morphine in the 
brain with time. These investigators administered 25 mg/kg codeine-N-I^CH by 
subcutaneous injection to rats. These rats were killed after 15, 30, 60 and 150 
minutes respectively. The spinal cord and the brain were removed, and the 



28 



concentrations of these drugs were determined in the spinal cord, hypothalamus, 
cerebellum, medulla oblongata, mid-brain and parts of the hemispheres. 

Miller and Elliott (1955) reported that concentrations of codeine rose 
sharply from the 15th to the 30th minute, while a slower rise was seen from the 
30th to the 60th minute. The highest concentration of codeine was measured 60 
minutes after drug administration. Another important fact to be considered is that 
relatively small amounts of both codeine and morphine enter the brain, and it is 
these concentrations which are responsible for producing analgesia. These 
results indicate a relationship between drug concentrations of codeine and 
morphine in the brain and the degree of analgesia produced. The exact 
relationship is yet to be firmly established. < ' * 

1.5 Genetic Polymorphism 

Genetic variation is an important cause of the large differences seen in 
drug metabolism between individuals. A number of isoenzymes in the 
cytochrome P450 family are involved in the oxidative metabolism of several 
essential drugs (Nebert et al., 1987). The entire population can be divided into 
extensive and poor hydroxylators based on the extent to which they metabolize 
certain drugs like mephenytoin, debrisoquine, sparteine and procainamide. It is 
well known that 0-demethylation of codeine, leading to the formation of the 
analgesically active metabolites morphine and morphine-6-glucuronide, is 
catalyzed by cytochrome P450 IID6 isoenzyme (Nebert et al., 1989) and 
cosegregates with the debrisoquine/sparteine oxidative polymorphism . 

Five to ten percent of Caucasians are classified as "poor metabolizers" 
(PMs), since they lack the specific isoenzyme for 0-demethylation (Yue et al., 



29 



1990 a, b), while the remainder, capable of 0-demethylating codeine, are termed 
"extensive metabolizers" (EMs). PMs are rare in the native Chinese population 
compared to Caucasians (Yue et al., 1989 a) indicating inter-ethnic differences in 
drug metabolism. This pharmacogenetic bias translates clinically into the fact 
that the PMs may not derive as much analgesic effect as the EMs. There are no 
data in the literature regarding genetic factors influencing the N-demethylation of 
codeine. 

Glucuronidation reactions are catalyzed by a group of isoenzymes with 
overlapping specificities. Glucuronidation of codeine has been shown to be 
induced by smoking and oral contraceptives (Yue et al., 1990 c). It is also 
affected by the co-administration of other drugs like diazepam and 
chloramphenicol. ; f; f ■'. j ; : ? ^ ' '-''"'- ' \ 

1.6 Immunomodulation 

The immune process consists of a number of concerted events which 
include recognition and processing of foreign antigens, proliferation and 
differentiation of responder cells, and the production of proteins and peptides for 
the amplification and mediation of the immune response. In this regard, the 
immune system is not isolated and autonomous in nature. In fact it is a part of an 
interactive and communicative triad, that also includes the nervous and 
endocrine systems. 

The immune system can affect brain functions as evidenced by the 
release of neurotransmitters and enhanced brain activity in certain localized 
regions after activation of the immune system following bacterial infection 
(Saphier, 1987). A number of centrally acting pharmacological agents have been 



30 



shown to affect immune function directly or indirectly via the hypothalamo- 
pituitary-adrenal (HPA) axis. Within this axis, feedback loops have been 
identified which, when activated by certain immune products such as cytokines, 
are responsible for the production of glucucorticoids, leading to marked 
immunomodulatory effects. These effects are enhanced in stressful situations 
(Herz, 1993). This is further indicative of the relationship between the brain and 
the immune system, either by neuronal pathways or by modulation of endocrine 
system 

Drugs of abuse, particularly opioids, can produce deleterious effects on 
the immune system. From an immunological standpoint, it is necessary to 
determine whether the effects of drug abuse were due to the drugs, or the 
consequence of needle sharing and poor nutrition--both of which are frequently 
observed factors associated with drug addiction. Earlier epidemiological findings 
suggested that increased infections were caused by sharing of unsterilized and 
contaminated needles. However, subsequent clinical studies have focused on 
the increasing evidence that the opioids themselves are capable of affecting the 
host defense mechanisms by directly acting on the immune system. 

1.6.1 Opioids Receptors 

The concept of opioids as immunomodulators is based on studies 
showing the presence of classical opioid receptors on the surface of cells of the 
immune system. Ovadia et al. (1989) demonstrated that rat lymphocytic 
membranes possess a certain GTP binding protein that couples opioid receptors 
to adenylate cyclase in response to the action of an opioid on the lymphocytes. 
Wybran et al. (1979) reported an opioid involvement in immune function based 



31 



on their observations with human T lymphocytes. They indicated that the opioid- 
induced suppression of lymphocytes was reversible with naloxone. 

Carr et al. (1994) found that naltrexone antagonized both the analgesic 
and immunosuppressive effects of mice, suggesting the involvement of the same 
receptor for both actions, that is, the |i receptor. This inference is supported by 
the findings of Ward et al. (1984) which showed that a ^-selective antagonist |3- 
funaltrexamine, and not the 5-selective antagonist naltrindole (Portoghese et al., 
1988) or the K-selective antagonist nor-binaltorphimine (Portoghese et al., 1987), 
attenuated morphine-induced immune suppression. 

1.6.2 Effects on Lymphocytes 

Lymphocytes are the primary immunocytes responsible for cell-mediated 
(T lymphocytes) as well as humoral (B lymphocytes) immunity as seen in Figure 
1-7. Measurement of the effect of various drugs on the proliferative capacity of 
lymphocytes is the most common in vitro assessment of functional cell-mediated 
responses. A large number of lymphocyte markers are suppressed following 
acute and/or chronic administration of opioids (Herz, 1993). 

Naloxone-reversible reduction in the number of circulating lymphocytes in 
morphine-treated rabbits has been reported along with effects on the various 
subpopulations of lymphocytes (Herz, 1993). Arora et al. (1990) found an 
increase in the T helper to T suppressor ratio following morphine treatment. T 
lymphocyte rosette formation was one of the first functional measures of T ceil 
function to be studied for comparing the immunosuppressive potential of various 
drugs. Wybran et al. (1979) showed that the suppressive effects of opioid 
agonists, in particular morphine, on the T cell rosette formation was 



32 



stereospecific for levorotatory forms, naloxone reversible, and produced 
tolerance. 

Humoral immunity, on the other hand, involves antibody responses to 
drugs which are recognized as antigens by the immune system. Primary 
antibody responses were seen to be suppressed by the action of opioids in 
sheep erythrocytes (Lefkowitz and Chiang, 1975). Plaque-forming responses, 
which are indicative of antibody production, were suppressed in splenocytes 
obtained from mice implanted with morphine pellets (Bryant et al., 1990). 

1.6.3 Effects on Myeloid Cells 

Cells of myeloid origin include monocytes, macrophages, neutrophils, 
mast cells, basophils and eosinophils. In addition to serving as mediators of 
inflammatory responses, they are involved in numerous functions critical to early 
immune response. These functions include antigen presentation, antibody 
production, lysis of tumor cells, phagocytosis of foreign particles and the release 
of immune response mediators such as interferons, cytokines, transforming 
growth factor (TGF) and tumor necrosis factor (TNF). 

In humans, morphine exposure leads to a depression of the phagocytic 
properties associated with myeloid cells (Tubaro et al., 1985). Macrophages, 
which are activated by the stimulatory agent y-interferon, were inhibited in 
morphine-pelleted mice and the tumoricidal activity of the macrophages was 
seen to be completely abolished (Bryant et al., 1988 a). Chronic morphine 
administration in mice also inhibited macrophage colony formation as the result 
of a decreased expression of the macrophage stimulating factor (Herz, 1993). 



33 




® 



s 
§ 

■a 

M 

I 



-li 






^••^ :■ 



■®i 



= i^D 



_ >: «> 6 




CO 



0) 

X 

E 
o 

»*— 

■a 

Q. 
CO 
T3 

JO 

"53 
o 

■g 

o 

JZ 
Q. 

E 



c 
q 

o 

c 

M— 

T3 

C 
CO 

c 
g 

CO 

o 

CO 
W) 
_CD 

o 

jr 
H 



CD 

13 
D) 
Li. 



34 



1 .6.4 Effects on Natural Killer Cells 

Natural killer cells represent another type of immune cell and constitute 
only about 5% of the total leukocyte population. They are large cells possessing 
an intrinsic activity for non-specific killing and lysis of a variety of tumor cells. In 
addition to serving as scavengers of malignant cells, natural killer cells also play 
a regulatory role in antibody production. 

Shavit et al. (1984) demonstrated that daily doses of subcutaneous 
morphine (50 mg/kg) suppressed natural killer cell tumoricidal activity (NK 
activity), and that naltrexone administration abolished this effect. This is strong 
evidence that the natural killer cytotoxic effect is centrally-mediated. Shavit et al. 
(1986 a) injected small quantities of morphine directly into the lateral ventricles 
and found that the NK activity was markedly suppressed. Novick et al. (1989) 
reported a marked decrease in NK activity in heroin addicts on methadone 
maintenance therapy. ' ' 

NK activity is usually below normal basal levels in HIV patients and 
declines as the disease progresses. Endogenous opiate peptides, leucine- 
enkephalin and methionine-enkephalin have been reported to increase the 
cytolytic capacity of the natural killer cells (Wybran et al., 1987 ; Oleson and 
Johnson, 1989). This indicates that such candidates can potentiate the NK cell 
responses and restore immunocompetence in the case of pre-AIDS and AIDS 
patients. 

1.6.5 Mechanism of Action 

Opioids have been shown to down-regulate immune responses. The 
exact mechanism by which this immunosuppression occurs has yet to be 



35 



established. A direct mechanism of action is thought to be through lymphocyte 
opioid receptors (Figure 1-8). The second hypothesis is that the immune effects 
are indirectly mediated, either by the activation of the hypothaiamic-adrenal- 
pituitary (HPA) axis with subsequent increase in the production of adrenal 
corticosteriods or by the release of catecholamines as a result of sympathetic 
innervation. 

It has been observed that systemic administration of morphine 
suppresses the activity of NK cells in the rat (Shavit et al., 1984, 1986 a, b). The 
same group also reported that central administration of morphine produced the 
similar results, but required doses one third of those administered systemically. 
The NK suppression was blocked by naltrexone. N-Methyl morphine, a morphine 
analog which cannot cross the blood brain barrier, had no effect on the NK 
cytotoxicity when administered systemically. This is further evidence that the 
immunosuppressive effects of morphine are mediated by opioid receptors in the 
brain. 

Morphine and other opioids are known activators of the HPA axis, and 
induce glucocorticoid output. Corticosterone has been implicated as possessing 
potential immunosuppressive effects as it was able to dose-dependently 
suppress NK activity in vitro. This effect was also observed in vivo in mice 
implanted with morphine pellets (Freier and Fuchs, 1994). A glucocorticoid 
receptor antagonist, RU 38486, blocked morphine-induced suppression of NK 
activity in a dose-dependent fashion. Naltrexone administration antagonized the 
morphine-induced elevation in serum corticosterone. This suggests that 
suppression of NK activity is linked to glucocorticoid elevation, which is the result 



3& 



MORPHINE 




O 



mMUNE SySTEM: boTs^ 

marrow, lymph nodes, thymus, 
spleen, somatic tissues, vascular 
space 



Figure 1-8 : The mechanisms of opioid-lnduced immunosuppression (from 
Peterson eta!., 1990). 



studies have shown that exposure of immune cells to opiates, especially 
morphine, results in a variety of functional disturbances (Chao et al., 1992, 1993; 
Peterson etal., 1991,1993). 

There are many reports in the literature implicating opiates as 
immunomodulatory agents. Most of these studies have focused on morphine, the 
narcotic of choice In case of severe pain associated with trauma and cancer, and 
have shown that morphine possesses potent immunosuppressant activity. There 
are, however, no studies that investigate the immunosuppressive effects of 
codeine or any of the glucuronide metabolites of codeine and morphine. 

A technique that simulates cellular immune response in vitro is the mixed 
lymphocyte reaction (MLR). It is based on the observation that lymphocytes from 
a mixture of genetically different individuals with different HLA (Human 
Leukocyte Antigen) types react with each other and proliferate. This test is 
performed by preventing the response of one set of lymphocytes (donor) through 



37 



radiation which then allows only the other set of lymphocytes (recipient) to 
proliferate as shown in Figure 1-9 . 




in 
in 

0) 

c 

c 
>, 
W 

< 

Z 
Q 



I I I I I I 
12 3 4 5 6 

Days 



Figure 1-9 : A mixed lymphocyte reaction with cell proliferation. 

Thus, opioids possess receptors which are capable of modifying immune 
functions. The concentrations achieved with analgesic doses of opioids are 
similar to those reported in the in vitro immunomodulatory experiments. It is also 
apparent that stress-induced activation of the endogenous opioid networks can 
contribute to various immunological changes. The focus for future research must 
include an understanding of the role of opioids in regulating immunity and their 
interaction with other immune function mediators. 



1.7 Receptor Binding 

In the case of morphinans, the aromatic ring and the basic nitrogen atom 
are necessary for analgesic activity. Substitutions at the phenolic hydroxyl group 
(position 3) and the alcoholic hydroxyl group (position 6) have been shown to 
cause pharmacological profiles which are opposite in nature. While additions at 



m 



the 6-position were seen to enhance opioid receptor binding affinities, changes 
at the 3-position significantly decreased receptor binding (Labella et a!., 1979). 
The structure activity relationships of morphine and its 3- and 6-glucuronide 
nnetabolites have been evaluated in several studies (Yaksh et al., 1986 ; Gong 
eta!., 1991 ; Shimomura et al., 1971 ; Pasternak et al., 1987). 

On the basis of these results, investigators speculated that the inactivity of 
morphine 3-glucuronide compared with morphine 6-giucuronide could be due to 
the differences in the level of receptor binding. Christensen and Jorgensen 
(1987) showed that morphine 6-glucuronide, but not morphine 3-glucuronide, 
had a high affinity for the opiate receptors isolated from bovine brains in 
competition with ^H-naioxone. Subsequent investigation by Pasternak et al. 
(1987) identified that morphine 6-glucuronide interacts with [i- but not k- 
receptors. 

The greater potency of morphine 6-glucuronide compared to morphine in 
antinociception studies has been reported by various investigators (Abbott and 
Palmour, 1988 ; Sullivan et al., 1989 ; Paul et al., 1989). In contrast to morphine 
and morphine 6-glucuronide analgesia, morphine 3-glucuronlde produces 
hyperalgesia, respiratory stimulation and behavioral excitation by non-opioid 
mechanisms (Yaksh et al., 1986 ; Pelligrino et al., 1989). These studies suggest 
that morphine 3-glucuronide can actually antagonize the effects of both 
morphine and morphine 6-glucuronide. This is clinically important not only for the 
analgesic effect, but also for the respiratory depression associated with morphine 
administration. 



39 



1.8 Hypotheses 

The hypotheses of this project are based on the overall aim of the project, 
that is, to examine and compare the analgesic and immunomodulatory effects of 
codeine and codeine 6-glucuronide. 

1. Codeine 6-glucuronide, like morphine 6-glucuronide, possesses 
analgesic activity. 

2. The glucuronide metabolites of codeine and morphine are less 
immunosuppressive than their parent compounds. 

1.9 Specific Objectives 

1. Develop and validate a reliable and sensitive HPLC-UV based assay 
for the quantitation of codeine, morphine, codeine 6-glucuronide, 
morphine 6-glucuronide and morphine 3-glucuronide in biological 
samples. -•. - , . ■. •■■■ . 

2. Chemically synthesize codeine 6-glucuronide utilizing a modification 
of the Koenigs-Knorr reaction. '^ 

3. Assess the immunomodulatory effects of codeine, morphine and their 
6-glucuronide metabolites in human T lymphocytes (in vitro). 

4. Compare the analgesic potencies of codeine and codeine 6-glucuronide 
in the rat using the tail flick method after intracerebroventricular (i.c.v.), 
subcutaneous (s.q.) and intravenous (i.v.) routes of administration. 

5. Determine the [i opioid receptor binding affinities of codeine and 
codeine 6-glucuronide. 

6. Analyze plasma and brain concentrations of codeine and their 



40 



metabolites at the peak analgesic response time after administration of 
codeine and codeine 6-glucuronide by various routes as described in 
specific objective #3. 



^ . * 



CHAPTER 2 
METHODS 

2.1 Specific Objective #1 : 
Analytical Method 

An isocratic HPLC method was developed using a ultraviolet absorbance 
detector along with an efficient solid phase extraction method to analyze 
physiological concentrations of codeine, codeine 6-glucuronide, morphine, 
morphine 6-glucuronide and morphine 3-glucuronide in various biological 
samples, that is, human urine, rat plasma and rat brain. The method was first 
developed using blank human urine. It was then validated using rat plasma and 
brain samples. 

2.1.1 Materials 

HPLC grade methanol and acetonitrile were used (Fischer Scientific, 
Fairlawn, NJ, USA). Analytical grade potassium dihydrogen phosphate and 85% 
v/v o-phosphoric acid were supplied by Sigma (St. Louis, MO, USA) as were 
codeine, morphine, morphine 6-glucuronide and morphine 3-glucuronide. A 
sample of codeine 6-glucuronide was donated by the National Institute on Drug 
Abuse (NIDA, Rockville, MD, USA). 

2.1.2 Extraction Procedure 

2.1.2.1 Human urine 

Solid phase extraction was performed with 3 ml Clean Screen® columns 
(Worldwide Monitoring, Horsham, CA, USA) containing 40 micron bonded silica 



41 



42 



particles. The cartridges were placed on a 12 station Vac-Elut (Varian, Harbor 
City, CA, USA). The columns were conditioned with methanol (3 ml), distilled 
water (3 ml) and 0.025 M phosphate buffer pH 3 (1 ml). A 1 ml sample of urine 
mixed with 2 ml of phosphate buffer pH 3 was then loaded onto the column. The 
cartridges were air-dried for 30 seconds and then washed with 0.025 M 
phosphate buffer pH 3 (1 ml), followed by methanol (1 ml). The columns were 
air-dried again for 30 seconds before eluting the compounds with 3 ml of 5% 
freshly prepared ammoniacal methanol solution. The eluent was evaporated to 
dryness under a stream of nitrogen gas and the residue was reconstituted in 150 
)Ltl of the mobile phase and 50 ^\ was injected into the HPLC system. 

2.1.2.2 Rat plasma 

Extractions were performed with 10 ml Clean Screen® columns. The 
columns were conditioned with methanol (10 ml), distilled water (10 ml) and 
0.025 M phosphate buffer pH 3 (2 ml). A 200 ^L sample of rat plasma was mixed 
with 400 nL of 0.025 M phosphate buffer pH 3 and loaded onto the column. The 
cartridges were air-dried for 30 seconds and then washed with 0.05 M acetate 
buffer pH 4.5 (2 ml), followed by methanol (2 ml). The columns were air-dried for 
30 seconds before eluting the compounds with 3 ml of 10% freshly prepared 
ammoniacal methanol solution. The eluent was evaporated to dryness under a 
stream of nitrogen gas. The residue was reconstituted in 150 ^\ of the mobile 
phase and 50 ^1 was injected into the HPLC system. 

2.1.2.3 Rat brain 

Brain samples were accurately weighed and an aliquot of 0.025 M 
phosphate buffer pH 3 (1 ml) was added to each sample. The samples were then 
homogenized and transferred to borosilicate tubes. After addition of a further 2 



m 



ml 0.025 M phosphate buffer pH 3, the samples were placed in a shaker for 10 
minutes. The samples were then centrifuged at 4000 rpm for 20 minutes. The 
supernatant was removed and loaded onto 10 ml Clean Screen® columns. The 
columns were conditioned with methanol (10 ml), distilled water (10 ml) and 
0.025 M phosphate buffer pH 3 (2 ml). After the samples were loaded, the 
cartridges were air-dried for 30 seconds and washed with 0.01 M acetate buffer 
pH 4.5 (2 ml) followed by methanol (2 ml). The columns were air-dried for 30 
seconds before eluting the compounds with 3 ml of 10% freshly prepared 
ammoniacal methanol solution. The eluent was evaporated to dryness under a 
stream of nitrogen gas. The residue was reconstituted in 150 ]x\ of the mobile 
phase and 50 \i\ was injected into the HPLC system. 

2.1.3 Chromatographic Conditions 

2.1.3.1 HPLC system 1 

This system was used to examine human urine samples for the presence 
of opiates. It consisted of a Waters 501 multi-solvent pump set at a flow rate of 
0.9 ml/min. The mobile phase consisted of 82% acetonitrile and 18% phosphate 
buffer (0.05 M potassium dihydrogen phosphate adjusted to a final pH of 3 with 
85% v/v o-phosphoric acid). Separation of the compounds was achieved on a 20 
cm X 4.5 mm I.D. Accubond® diol column with a 5 micron particle size (J & W 
Scientific Inc., Folsam, CA, USA). A Spectra-Physics Focus® multiwavelength 
forward optical scanning detector (San Jose, CA) set at 220, 230 and 280 nm 
was used to detect eluting compounds. Data acquisition and analysis was 
performed with Autolab® software loaded on a 386 IBM computer. 



44 



Apart from chromatographic analysis, the Autolab software provided the 
option to examine the UV spectra of the compounds in the chromatograms. All of 
the compounds of interest exhibited a maxima in their UV spectra around 285 
nm. A specific opiate of interest could be further characterized using derivative 
spectroscopy. In derivative spectroscopy, the first or higher derivative of 
absorbance with respect to wavelength is recorded versus wavelength. In this 
way the ability to detect and measure minor spectral features is considerably 
enhanced (Willard, 1986). 

2.1.3.2 HPLC system 2 

This system was used to determine concentrations of codeine, morphine 
and their glucuronides in rat plasma and brain samples. It consisted of a Waters 
501 multi-solvent pump set at a flow rate of 0.7 ml/min. The mobile phase 
consisted of 88% acetonitrile and 12% phosphate buffer (0.05 M potassium 
dihydrogen phosphate adjusted to a final pH of 3 with 85% v/v o-phosphoric 
acid). Separation of the compounds was achieved on a 20 cm x 4.5 mm I.D. 
Accubond® diol column with a 5 micron particle size (J & W Scientific Inc., 
Folsam, CA, USA). Sample injection was automated by the use of a Waters™ 
717 Plus autosampler. A Waters 486 tunable absorbance detector set at 220 nm 
was used to detect eluting compounds. A Millenium® 2010 Chromatography 
Manager software was used to acquire data. All the instruments were controlled 
by a 386 NEC Powermate computer, which also stored and processed the 
acquired chromatographic data. V' 



45 



2.2 Specific Objective #2: 
Synthesis of Codeine 6-glucuronide 

Codeine 6-glucuronide is not available commercially. In order to evaluate 
its pharmacological activity in the rat, relatively large amounts of this compound 
(100-200 mg) were required. The National Institute of Drug Abuse (NIDA) is the 
only agency which provides samples of this compound, basically for analytical 
purposes (5 mg). For this reason, it was decided to synthesize codeine 6- 
glucuronide using a reproducible method described in the literature (Yoshimura 
et al., 1968). The initial attempt to synthesize codeine 6-glucuronide with some 
modifications of the Koenigs-Knorr reaction provided a good yield of the product, 
comparable to that reported in literature. A scheme of the synthetic route (Figure 
2-1) is shown below. 

2.2.1 Reaction Step I 

In the first step, codeine monohydrate (1 g) was dissolved in 200 ml of dry 
benzene in a three-necked flask attached to a condenser with a drying tube 
containing drierite (CaS04) and a Dean-Starke trap. The trap was used to 
periodically distill benzene and to maintain anhydrous conditions in the flask. The 
solution was heated at 150 °C with an oil bath. Small amounts (250 mg) of 
freshly prepared silver carbonate along with equal portions of a solution 
containing 5 g of the acetobromo derivative of glucuronic acid, that is, methyl 
2,3,4-tri-0-acetyl-1-bromo-1-deoxy-D-glucupyranuronate (obtained from NBS 
Biologicals Inc., Herts, UK), in 100 ml of dry benzene were added every hour 
over two seven hour periods. 



46 



MeOOC_Q Br 



^i^ (Protected Glucuronic Acid) 

~^ " // 

Silver Carbonate ^ 

\ in Benzene Me 




Codeine 





)Ac 

"Intermediate" 



Sodium Methoxide 
in Methanol 

Me 



1 . Barium Hydroxide 



2. Oxalic Acid 



Codeine 6-glucuronide 




Figure 2-1 : Synthetic route of codeine-6-glucuronide using the Koenigs-Knorr 
reaction (adapted from Yoshimura et a!., 1968). 



Thin layer chromatography (TLC) was performed at regular intervals 
during the heating period to monitor the extent of the reaction. The solvent used 
for the TLC was a mixture of methanol and methylene chloride (1:4). Small 
portions (1 ^1) were removed from the boiling mixture and spotted on TLC plates 
(Brinkman Polygram Sil G/UV 254). With increasing time, the spot representing 
the starting material codeine, disappeared and another spot with a higher Rf 
value appeared. This indicated the formation of a product assumed to be methyl 
[codein-6-yl 2,3,4-tri-0-acetyl-1-bromo-1-deoxy-D-glucupyranosiduronate, that is, 
the 6-glucuronide of codeine with intact acetyl and methyl groups on the 
glucuronic acid moiety, and subsequently referred to as the intermediate. The 



47 



structure and identity of the compound in CDCI3 was confirmed by H NMR 
(Varian EM 390 Spectrometer). 

After the heating was stopped, the contents of the flask were filtered and 
the clear filtrate was evaporated to dryness with a rotory evaporator. The residue 
was redissolved in absolute ethanol and evaporated to dryness. The solid 
residue was then transferred to a column and chromatographed using 500 ml 
each of ethyl acetate, ethyl acetate : ethanol (60 : 40), ethanol : methanol (50 : 
50) and methanol. Various fractions were collected and concentrated to dryness. 
The dried fractions which indicated the presence of the compound of interest 
were recrystallized using methanol. The melting point of the compound was 113- 
116°C, in agreement with the value previously reported (Yoshimura et a!., 1968) 

2.2.1.1 Dry benzene 

A key point to guarantee the success of the first step was to ensure that 
the benzene used was absolutely dry. This was achieved by boiling benzene in 
the same apparatus as the reaction was done and removing the water with the 
Dean-Starke trap. Boiling for about 4 hours ensured removal of all the water 
present in the benzene. 

2.2.1.2 Fresh silver carbonate 

Freshly prepared silver carbonate is another essential prerequisite in the 
first step of the reaction, as the silver carbonate used as a catalyst is susceptible 
to oxidation in the presence of moisture and light. The procedure for preparing 
"active" silver carbonate (Wolfrom and Lineback, 1963) involves adding an 
aqueous solution of anhydrous sodium carbonate (1.6 g in 7.5 ml of distilled 
water) dropwise into a mechanically stirred solution of 8 g of silver nitrate 
dissolved in 20 ml of water. A solution of 1 g of anhydrous sodium hydrogen 



48 



carbonate in 12.5 ml of water was then added to the above in 2-3 portions. The 
mixture foams and a yellowish precipitate is formed. The solid recovered by 
filtration was silver carbonate. 

2.2.2 Reaction Step II 

The intermediate compound from step I (0.6 g) was suspended in a test 
tube with 3.5 ml of absolute methanol. A 1% solution of sodium methoxide in 
methanol (2 ml) was added and the mixture was stirred with a magnetic stirrer. 
The solution was evaporated to dryness in vacuo. 

2.2.3 Reaction Step III 

The dried residue from step II was dissolved in 2.2 ml of a 0.43N Ba(0H)2 
solution and stirred for about 4 hours before leaving it overnight in a refrigerator 
at 4° C. The barium salt which precipitated on cooling was dissolved in 4 ml of 
distilled water and adjusted to pH 6 with 2N oxalic acid solution. The solution 
was refrigerated overnight and the barium oxalate formed was removed by 
filtration. The filtrate was evaporated to dryness and the residue was 
recrystallized from methanol. The compound decomposed at 225-230 °C, which 
was slightly lower than that reported in the reference paper. 

2.3 Specific Objective #3: 
Analgesic Activities of Codeine and Codeine 6-glucuronide 

The polar glucuronide metabolites of codeine and morphine do not cross 
the blood brain barrier (BBB) as efficiently as their parent compounds. With this 
in mind, initial studies were designed to bypass the BBB and determine if 



48 



codeine 6-glucuronicle produced analgesia in pain-induced rats by using 
standard antinociceptive tests, that is, tail flick and hot plate methods. This was 
done by delivering the compounds directly Into the brain of rats via the 
intracerebroventricular route (i.c.v.). The next step was to compare the activities 
of codeine and codeine 6-glucuronide after subcutaneous and intravenous 
administrations using the same methods as in the i.c.v. studies for measuring the 
effects. 

During the synthetic procedure, the intermediate compound formed after 
the first reaction step was isolated and characterized. This intermediate is the 
glucuronic acid moiety attached to the 6-position of codeine with intact 
acetyl/methyl groups. The antinociceptive activity of this intermediate was also 
investigated. 

2.3.1. Intracerebroventricular Route Studies 

These studies involved investigation of the antinociceptive effects of 
codeine, codeine 6-glucuronide and the Intermediate compared to the standard 
analgesic drug, morphine. All drugs were dissolved In physiological saline (pH 
4.5 - 5.5) and administered at doses of 100 [jg/5 pi for codeine, 10 pg/5 pi for 
codeine 6-glucuronide and the intermediate, and a dose of 5 pg/5 pi for 
morphine. Each compound was administered to groups of 6 rats. One group of 
rats was injected with saline only and served as a control group. Another group 
was not Injected with anything to control for any possible effects of the surgical 
procedure on the pharmacodynamic measurements. Measurements were made 
at the following time points after administration of the compounds : 0, 10, 20, 30, 
40, 60, 90, 120, 150 and 180 minutes. 



so 



2.3.1 .1 Surgery 

After the animals were acquired, they were housed in cages in a room 
with a 12 hour light-dark cycle. The animals were fed on standard laboratory rat 
chow and tap water ad libitum. Each rat was anesthetized with 30-50 mg/kg 
sodium pentobarbital intraperitoneally and stereotaxically fitted with a 23 gauge 
intracerebroventricular stainless steel guide cannula. The co-ordinates used for 
the stereotaxic apparatus were : 1.0 mm lateral; 1.0 mm caudal to the bregma 
and 5.0 mm below the skull surface (Paxinos and Watson, 1986). The rats were 
allowed to recover for 3-5 days prior to starting the antinociceptive experiments. 

2.3.1.2 Tail flick method 

Analgesia was determined using the tail flick method of Berglund and 
Simpkins (1988) and previously described in section 1.4. Reduction in pain was 
expressed as the% of the maximum possible effect (% MPE) calculated as : 



o/o MPE = ( ^QSt latency - baseline latency ) , ^^^ 
( cut-off period - baseline latency ) 



The area under the effect curve (AUEC) was determined from individual% 
MPE versus time graphs using trapezoidal calculation. The AUEC is considered 
a good indicator for comparing the intensity of effect during a certain time period. 
It also provides a good estimate of the duration of effect produced by each 
compound. Comparison of total AUEC values are, therefore, considered to 
reflect the relative effectiveness of the compounds. 



51 



2.3.2 Subcutaneous Route Studies 

After the intracerebroventricular route, studies were performed by injecting 
the test compounds subcutaneously, that is, under the nape of the neck. 
Codeine, codeine 6-glucuronide and the intermediate were dissolved in 
physiological saline (pH 4.5 - 5.5) and injected at a dose of 10 mg/kg. A higher 
dose of 20 mg/kg of codeine was also administered to a group of 6 rats. The 
volume of injection was 1 ml/kg body weight of the rat. Each rat was also 
administered saline in a separate study and therefore, acted as its own control. 

Assessment of the reduction in pain was determined by the tail flick 
method as described for the intracerebroventricular route. The intervals between 
time points for measuring the response were, however, greater than in the 
intracerebroventricular studies so as to take the absorption factor into 
consideration. The measurement time points were 0, 15, 30, 45, 60, 90, 120, 
180, 240, 300 and 360 minutes. 

2.3.3 Intravenous Route Studies 

In these studies, the jugular vein of rats was catheterized so that 
compounds could be directly injected into the general blood circulation. Animals 
were first anesthetized with ketamine/xylazine (45:9 mg/100 g body weight of rat) 
intraperitoneally. A heparinized (100 U/ml) catheter (PE50 tubing, 0.58 mm X 
0.965 mm) was then placed in the right jugular vein. The catheters were 
stoppered and exteriorized between the scapulae to avoid chewing. The rats 
were allowed 1-2 days to recover from the anesthesia and surgery. Drugs were 
dissolved in physiological saline (pH 4.5 - 5.5) and injected through the catheter. 
Codeine, codeine 6-glucuronide and the intermediate were administered at a 



., "■» 



52 

.... /'\ 



dose of 10 mg/kg. The injection volume was 1 mg/kg body weight of the rat. 
Physiological saline (500 |il) was also injected to ensure that the drug reached 
the systemic circulation and did not remain in the dead volume of the catheter. 

As in the intracerebroventricular and subcutaneous studies, the tail flick 
method was used to determine the analgesic effect. As there was no absorption 
phenomenon to consider, periods between measurement times were less than in 
the subcutaneous studies but greater than the intracerebroventricular studies, 
that is, 0, 10, 20, 30, 40, 60, 90, 120, 180, 240 and 300 minutes. 

2.3.4 Statistics 

The area under the effect curve data for each compound was compared 
to its saline treatment using a paired Student's t-test. The area under the effect 
curve (AUEC) of compounds were also compared with each other using an 
unpaired Student's t-test. 

2.4 Specific Objective #4: 
Immune Studies with Human T Lymphocytes 

In vitro immune studies on the drugs were performed with human T 
lymphocytes found in peripheral blood mononuclear cells (PBMCs) which were 
isolated from the blood of healthy volunteers using the Ficoll-Hypaque density 
gradient centrifugation procedure. The isolated T lymphocytes were then 
stimulated by mitogens phytohemagglutin (PHA) and phorbol 12-myristate-13- 
acetate (PMA), activating the resting T cells and enabling them to become 
transformed into lymphoblast cells. These transformed cells are then capable of 
synthesizing DNA, dividing rapidly and proliferating. 



ii- 



53 



When the lymphocytes reached their peak proliferation, they were labeled 
with 1 pCi ^H thymidine after a period of 48 hours (for the PHA/PMA assay) and 
120 hours (for the mixed lymphocyte reaction assay). The cells incorporated the 
radiolabeled thymidine into their DNA and counts were done using a scintillation 
counter. The differences in cell count determined with and without drugs was 
used as a measure of drug-induced changes in lymphocyte proliferation. 
Immunosuppression produced by drugs was expressed in terms of% inhibition of 
proliferation and calculated as : . 



„. . uux- rr^ ir x- (CPM wjthout clf UQ - CPM wJth df uq) ^ ^ „- 
% Inhibition of Proliferation = -^^ ^ * 1 00 



(CPM without drug) 



2.4.1 Method 

-' i ;■■■' 
6 "< 

Peripheral blood (20 ml) was obtained from a healthy volunteer and 
transferred to a conical tube. To this conical tube 20 ml of RPMI tissue culture 
media containing 5% albumin was added (this culture media was developed at 
Rosewell Park Memorial Institute and hence the name, RPMI). A 10 ml Ficoll- 
Hypaque solution was drawn up in a pipette and carefully delivered to the bottom 
of the tube. The tube was then placed in a balanced centrifuge for 30 minutes at 
1200 rpm. A white layer containing lymphocytes formed between the culture 
media and red cells. The lymphocyte cells were transferred into another conical 
tube and diluted with 50 ml of the RPMI cell culture media. The tube was 
centrifuged again for 10 minutes at 1200 rpm. A "pellet" of cells was seen to be 
formed at the bottom of the tube. The supernatant was decanted until 1 ml of the 



54 



media remained in the tube with the pellet. The tube was then slightly agitated to 
disperse the pellet homogeneously in the media and a cell-count was done to 
determine the total number of cells/ml using tryphan blue stain under a light 
microscope (Fischer Scientific, Fairlawn, NJ, USA) at a magnification of 40 X. 
The cell suspension was diluted with RPMI media to obtain a cell-count of 1x 10^ 
cells/ml for PMA and PHA assays and 2x10^ cells/ml for the MLR assay. 

Stock solutions and dilutions of the drug to be tested were made in RPMI 
cell culture media. A 96-well U-bottom cell plate was used and 100 pi drug 
solutions of each concentration of each drug was pipetted into the plate in 
triplicate. To each drug-containing well was added, 100 pi of either PHA (5 
pg/ml) or PMA (50 ng/ml) containing T lymphocytes. The controls in the assay 
consisted of T cells without the drug, T cells containing the mitogens (PHA or 
PMA) but without the drug, and a control with only the cell culture media. The cell 
plate was covered on the top and placed in an incubator until it was radiolabeled. 

Standard drug dilutions were done in the same way as in the case of 
PHA/PMA assays. Lymphocytes from two separate individuals were used for this 
assay. The cells of one individual was selected to be irradiated and labeled as 

137 

the donor. This was done in a cell irradiator with a Cs source at 2500 rads for 
4 minutes. To 100 pi of drug solution of varying concentrations, 50 pi of the 
irradiated cells and 50 pi of the plain cells from the other individual were added. 
The cell plate was covered and incubated until it was radiolabeled. 

The cell plates in the PHA/PMA assay (on day 2) and MLR assay (day 5) 
were taken out of the incubator and placed on the work surface. ^H-Thymidine (1 
pCi) diluted with the RPMI culture media was taken out of the refrigerator. Using 



55 



a Hamilton microsyringe stored in ethanol, each cell well was given 10 |j| of the 
^H-thymidine solution. . - 

Cells were harvested onto nitromethylcellulose paper via a cell harvester 
attached to a vacuum pump. The cell plate was discarded in a radioactive waste 
box. Scintillation vials were set up and labeled appropriately. After the paper had 
dried, forceps were used to punch out individual circles (each representing a cell 
well) which were placed into the corresponding scintillation vials. Scintillation 
fluid (3 ml) was then added to each vial and the vials were capped tightly. 
Radioactivity counts were determined using a scintillation counter. 

2.4.2 Statistics 

The% inhibition of proliferation data of the compounds were compared to 
each other using an unpaired Student's t-test. 

2.5 Specific Objective #5: 
Receptor Binding Studies 

2.5.1 Materials 

Tris-HCI, bovine serum albumin (BSA), morphine sulfate and naloxone 
HCI were obtained from Sigma Chemicals (St. Louis, MO, USA). Sodium chloride 
was purchased from Fischer Scientific Inc. (Fairlawn, NJ, USA). Codeine 
phosphate was obtained from Westlab Pharmacy, Gainesville, PL. Codeine 6- 
glucuronide and the intermediate were synthesized using the Keonigs-Knorr 
reaction. ^H-DAGO ([ D-ala^ N-methyl-phe^ glyol^ ][ tyrosyl-3,5-^H ] enkephalin) 
was used as a competitive ligand for the binding studies and was purchased 
from Amersham (Arlington Heights, IL). 






2.5.2 Method 

Receptor binding studies were carried out using brain tissues from 
Sprague-Dawley rats (200-300 g) using a modification of the procedure reported 
by Hochhaus et al. (1988). Brain tissue was homogenized in 60 volumes of 50 
mM Tris-HC! buffer (pH 7.4) containing 100 mM sodium chloride. The 
homogenate was incubated in a water bath for 1 hour at 20 °C. After incubation, 
the homogenate was centrifuged at 20,000 rpm for 20 minutes at 4 °C. The pellet 
formed at the bottom was washed twice with 50 mM Ths-HCI buffer. The 
membrane suspension was vortexed for 2 minutes to ensure homogeneity. 

Competitive binding studies were performed after adding 1% w/v of 
bovine serum albumin (BSA) to the membrane suspensions. The suspension 
(880 ^l) was placed in polyethylene tubes and incubated with 20 |il of the tracer 
(1 nM ^H-DAGO, a |i-receptor selective agonist) and 100 |al of the competing 
ligand (various concentrations of the drugs dissolved in 50 mM TrIs-HCI buffer). 
This mixture was incubated in a water bath for 1 hour at 20 °C. After incubation, 
the bound and unbound fractions were separated by filtration using Whatman 
GF/C filters. The filter paper containing the retained radioactivity was transferred 
to scintillation vials and 3 ml of scintillation fluid was added to each vial. The vials 
were capped tightly and shaken to enable the radioactivity to be distributed in the 
scintillation cocktail. The vials were left overnight and tritium counts were 
performed using a liquid scintillation counter. 

Specific binding of morphine, codeine, codeine 6-glucuronide and the 
intermediate to the )a-receptor was determined by competitive displacement of 
the radiolabeled tracer by various concentrations of the test compounds. The 
non-specific binding (NS) was determined with a relatively high concentration of 



57 



morphine (1 |iM). The total binding (T) was determined from vials in which no 
drug was added. The counts per minute (CPMs) obtained from a scintillation 
counter were then plotted versus increasing drug concentrations. The data was 
fitted using an E^ax model from the Scientist program (Micromath Scientific Inc., 
Salt Lake City, UT) as described in Equation 1. The raw data (CPMs) for each 
compound were standardized by transforming them into% of total binding and 
plotting them as a function of increasing drug concentration (Equation 2). The 
equations used for fitting the data are as follows : 

Equation 1 : 



CPM = T - ^ + NS 



Equation 2 



CPM 

% Total Binding = * 100 



2.6 Specific Objective #6: 
Plasma and Brain Concentrations 



The objective of this study was to determine plasma and brain 
concentrations at the peak analgesic response time in the rat. This was achieved 



98 



by the administration of codeine or codeine 6-glucuronide to groups of rats (n=6) 
by intracerebroventricular, subcutaneous and intravenous routes (described in 
section 2.3). The peak response time from the tail flick experiments after each 
route of administration was used as the time at which plasma and brain samples 
were collected. 

Rats were first anesthetized with metofane 5 minutes prior to the peak 
response time. At the appropriate time, the rats were decapitated using a 
guillotine. Trunk blood samples were collected in vacutainers containing EDTA 
as an anticoagulant. Plasma was obtained by centrifuging the blood at 2500 rpm 
for 20 minutes. Brain samples were obtained after removing the skull and other 
membranes attached to the brain. Both the plasma and brain samples were 
stored in a freezer at -80 °C. The samples were analyzed using the HPLC 
method described in section 2.1. 



'•> r 



U .J 



r-J. Tv;- ■>■ 



CHAPTER 3 
RESULTS 



3.1 HPLC Development 

An HPLC-UV based method was successfully developed for the analysis 
of codeine, morphine, codeine 6-glucuronide, morphine 6-glucuronide and 
morphine 3- glucuronide in biological samples, that is, human urine, rat plasma 
and rat brain. A typical chromatogram obtained after injection of a standard 
solution containing 100 ng/ml of each compound is represented in Figure 3-1 
(system 1). This system uses a multiwavelength scanning UV detector and 
allows the simultaneous examination of the same chromatogram at different 
wavelengths in the scanning range (Figure 3-2). 



0.0100 1 
0.0080 - 

a 0.0060 

I 
g> 

5 0.0039 - 

< 

0.0019 
-0.0001 



M 



JU 



C60 



MCO 




\ I =T ^T 

8.00 11.20 14.40 17.60 

■nme(min) 



KOO 




-I —n- 

20.80 



^' 



24.00 



Figure 3-1 : A standard solution containing 100 ng/ml of each of all the 
compounds using system 1 . 



59 



0.0030 -, 

.| 0.0024 -I 

J 0.0018 • 
S 0.0012 

0.0006 - 



0.0000 




8.00 



20.80 



24.00 



Time ( min) 



Figure 3-2 : The same chromatogram as in Figure 3-1 showing the 
multiwavelength capabilities of the detector. 

The software also allows examination of the UV spectra at any time point 
in the acquired chromatogram (Figure 3-3). This can be used to identify the 
presence of an opiate, since all opiates exhibit a UV maxima of 285 nm. The 
differential UV spectra further helps to confirm the identity an opiate of interest 
(Figure 3-4). This is done by following minute changes in the spectra of an opiate 
and matching it with the spectra obtained from a standard solution of the same 
opiate. ' 



0.1000 



0.0800 



C 0.0600 

3 

WO 

u. 



15.527 



< 



0.0400 



0.0200 



0.0000 




240 



280 



320 



Wavelength (X) 

Figure 3-3 : A typical UV spectra which is exhibited by all opiates. 



61 



N ) 



15.527 15.527" 



Wavelength ( X ) 
Figure 3-4 : A normal (— ) and second derivative (— ) spectra of morphine. 

3.1.1 Extraction Recoveries 

Extraction recoveries for eacii compound were determined by comparing 
the peak area of an extracted standard to an unextracted standard. The elution 
solvent of methylene chloride-isopropanol-ammonium hydroxide recommended 
by Worldwide Monitohng for opiates gave good recoveries for codeine and 
morphine only, but not for the glucuronide metabolites. However, the use of a 
5% ammonium hydroxide solution in methanol enabled the efficiency of 
extraction of polar glucuronides from human urine samples to be increased. This 
elution solvent gave clean extracts and excellent recoveries in excess of 80% for 
all the compounds of interest. The% extraction recoveries for the various 
compounds of interest in human urine are represented in Table 3-1. 

3.1 .2 Ranee / Linearity of Standard Curve 

After the initial development of the chromatographic system and extraction 
procedure, calibration curves were prepared in drug-free human plasma and 



62 



urine. Standards were set up for each compound of interest in the biological 
matrix (plasma/urine) at concentrations of 0, 10, 50, 100, 200, 400 and 500 
ng/ml. Each point on the calibration curve was taken as the average of two 
determinations. The standard curve was determined from calibrators by the 
linear least squares fit to the equation y = mx + b, where x = concentration, y = 
drug peak area, m = slope of the line and b = y-intercept of the line. Linear 
correlations between the area under the peak and concentration of each 
compound was performed by regression analysis using the Microsoft Excel 
program. It was seen that each compound of interest had a good correlation with 
an R^ of 0.99 or better over the range 0-500 ng/ml (Figures 3-5 and 3-6). 



Compound 


% Extraction recovery ± sd (n=4) 


Codeine 


92 ±6 


Morphine 


90 ±8 


Codeine 6-glucuronide 


86 ±4 


Morphine 6-glucuronide 


85 ±8 


Morphine 3-glucuronide 


83 ±6 



Table 3-1 :% extraction recoveries for compounds of interest in human 
urine. 



3.1.3 Specificitv 



Drug-free plasma and urine samples were analyzed with and without the 
drugs. This was performed to show that the drug peaks were well separated from 



63 



the solvent front and did not have any interference from endogenous materials 
also eluting from the column. 

3.1.4 Sensitivity/Limit of Detection a nd Quantitation 

The detection limit was determined as the concentration of the sample 
which corresponds to the signal that is twice that of the baseline noise. When 
extracted from human urine using Clean Screen® columns, the minimum 
quantifiable concentrations of the vanous compounds using system 1 were : 5 
ng/ml for codeine and morphine; 10 ng/ml for codeine 6-glucuronide, morphine 
6-glucuronide and morphine 3-glucuronide. 

3.1.5 Precision and Accuracv 

Precision is defined as a measure of the closeness between replicate 
concentrations of a sample and its mean value. It is expressed as the% relative 
standard deviation about the mean (standard deviation/mean *100). Replicate (n 
= 7) analysis of the samples were done on the same day (intra-day) and over a 
period of two weeks (inter-day). Both intra-day and inter-day precision were 
determined to be less than 10% for all the compounds. The intra-day and inter- 
day precision values for codeine are shown in Table 3-2. Accuracy is expressed 
as the difference between the actual value and the mean measured value for 
each concentration. Accuracy determined from calibration graphs was less than 
10%. 



64 



n 
9> 



IS 

D. 



350^ 



300.. 



250.. 



200.. 



150.. 



100.. 



50.. 




. Morphine i 
.Codeine i 



# ,,.| „ ,,|.i,.| MM |i m < m i | iiii|i m | m i|iiiil 

50 100 150 200 250 300 350 400 450 500 
Concentration (ng/ml) 



Figure 3-5 : A typical calibration curve for codeine and morphine 
in spiked urine. 



9> 



n 

0) 

a. 




_»_M6G 
_ci_M3G 
_A-C6G 



|..l.|llll|llll|llll|....|l H l| 



50 100 150 200 250 300 350 400 450 500 
Concentration (ng/ml) 



Figure 3-6 : A typical calibration curve for codeine 6-glucuronide, 
morphine 6-glucuronide and morphine 3-glucuronide in spiked urine. 



m 



3.1.6 stability 

Stability of extracted plasma, urine and brain sannples were determined 
over a 24 hour period by replicate analyses during the assay development and 
validation. The variation in peak areas of samples determined from replicate 
analysis was less than 10%. 



Concentration 
( ng/ml ) 


Intra-day variability 
(%) 


Inter-day variability 
(%) 


10 


6.4 , , 


9.7 


50 


3.3 


5.8 


100 


4.1 


5.3 


200 


4.3 


6.7 


400 


4.5 


7.2 


500 


6.2 


8.7 



Table 3-2 : Intra-day and inter-day variability from replicate analysis of 
standards containing various concentrations of codeine. 

3.2 Synthesis 



In the first step of the synthetic procedure, the key to the success of the 
reaction was maintaining anhydrous conditions in the flask. This was achieved 
by using a Dean-Starke trap, which allowed distillation of benzene and water at 
regular intervals. The completion of the reaction was determined by the 
disappearance of the starting material by thin layer chromatography. The 
structure and identity of the compound formed after the first step was confirmed 



66 



1 ' . ■ . 

by H NMR. It indicated the presence of acetyl and methyl groups and the 

attachment of a glucuronic acid group to codeine. The melting point of the 

compound was 113-116°C in agreement with the value reported by Yoshimura et 

al. (1 968). The yield of this reaction was 70%. 

Codeine 6-glucuronide was recrystallized from methanol and decomposed 

at 225-230 °C, slightly lower than the value reported by Yoshimura et al. (1968). 

This may be due to the fact that the product was apparently anhydrous 

compared to the half-hydrate product reported in the reference paper. Absolute 

methanol was used to recrystallize the compound instead of a water-methanol 

mixture used in the reference. The identity of the product was confirmed by 

comparing the retention time of the chromatographic peak to an analytical 

standard by using the HPLC method previously described (section 2.1). The 

overall yield of the reaction was about 16%. 

3.3 Analgesia Studies 

3.3.1 Intracerebroventricular Route 

Intracerebroventricular administration of morphine, codeine, codeine 6- 
glucuronide and the intermediate produced significant antinociceptive responses 
in the rats. All the compounds tested produced a peak response about 20 
minutes after administration. Each rat was also administered saline which 
produced minimal changes to baseline responses. The data set for the effect of 
surgery on the response time and the analgesic responses are summarized in 
Tables 3-3 and 3-4, respectively. It was observed that the surgical procedure 



67 



itself had no effect on the response time (Figure 3-7) and the analgesic response 
(Figure 3-8) and over a 3 hour period. 



Tlme(mln) 


1 


2 


3 


4 


5 


Mean 


SD 


SEM 





11.8 


8.7 


8.1 


7.8 


8.4 


9.0 


1.6 


0.7 


10 


11.5 


6.8 


9.5 


11.2 


8.1 


9.4 


2.0 


0.9 


20 


9.2 


7.0 


10.3 


9.6 


10.3 


9.3 


1.4 


0.6 


30 


7.2 


8.5 


8.2 


11.1 


11.8 


9.4 


2.0 


0.9 


40 


7.4 


9.4 


9.6 


8.8 


11.3 


9.3 


1.4 


0.6 


60 


12.2 


12.2 


11.4 


9.6 


10.8 


11.2 


1.1 


0.5 


90 


9.7 


9.1 


10.3 


9.3 


11.1 


9.9 


0.8 


0.4 


120 


9.0 


11.2 


10.8 


8.4 


9.4 


9.8 


1.2 


0.5 


150 


10.8 


9.6 


11.8 


8.7 


9.7 


10.1 


1.2 


0.5 


180 


11.3 


9.2 


11.6 


9.2 


10.1 


10.3 


1.1 


0.5 



Table 3-3 : The data for the effect of intracerebroventricular 
surgery on the response time (in seconds). 



68 



Time(min) 


1 


2 


3 


4 


5 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


-1.1 


-6.1 


4.4 


10.6 


-0.9 


1.4 


6.3 


2.8 


20 


-9.2 


-5.4 


6.9 


5.6 


6.0 


0.8 


7.5 


3.4 


30 


-16.3 


-0.6 


0.3 


10.2 


10.8 


0.9 


11.0 


4.9 


40 


-15.6 


2.2 


4.7 


3.1 


9.2 


0.7 


9.5 


4.3 


60 


1.4 


11.2 


10.3 


5.6 


7.6 


7.2 


3.9 


1.8 


90 


-7.4 


1.3 


6.9 


4.7 


8.5 


2.8 


6.3 


2.8 


120 


-9.9 


8.0 


8.5 


1.9 


3.2 


2.3 


7.4 


3.3 


150 


-3.5 


2.9 


11.6 


2.8 


4.1 


3.6 


5.4 


2.4 


180 


-1.8 


1.6 


11.0 


4.3 


5.4 


4.1 


4.7 


2.1 



Table 3-4 : The data for the effect of intracerebroventricular 
surgery on the analgesic responses (in% MPE). 





69 






40 

^ 30 
u 

0) 

42. 

0) 

.i 20 

H 

d 

(A 

o 

0^ 10 

. 


.V 

• I I I I^-^ ^ — I i i 




"II ^ 






30 60 90 120 150 180 

Time (min) 




Figure 3-7 : The effect of the intracerebroventricular surgical procedure 
on the response time. 




100- 
90^ 

*- 8o;. 

u 

"J 6o;. 
1 ^°:- 

X 40.. 

re 

S 30.. 

**. 2o:. 

10 !. 

n 


r r T T^^ 1 1 1 




30 60 90 120 150 180 

Time(min) 


Fig 
on 


ure 3-8 : The effect of the intracerebroventricular surgical proc 
the analgesic response. 


edure 



70 



The response time (Tables 3-5 to 3-9) for all the treatments was 
transformed into% of maximum effect data (Tables 3-10 to 3-14). The average 
values from the data sets were then plotted as a function of time (Figures 3-9 
and 3-10). Morphine was the most effective of the compounds, producing a 
maximal suppression of pain, that is, 100% of the maximum possible effect 
(MPE) in the rats. Codeine 6-glucuronide and the intermediate also exhibited 
marked increases in antinociceptive responses, that is, up to 89 ± 6 and 81 + 9% 
of MPE (± standard error of the mean or SEM), respectively. Codeine also 
produced analgesia, with a peak effect of 63 ± 2% of MPE. The% MPE at peak 
response time for all the compounds is summarized in Table 3-15. The area 
under the effect curve (AUEC) was determined from individual% MPE versus 
time graphs for each treatment using trapezoidal calculation (Figure 3-11) and is 
summarized in (Table 3-16). < , , -,-<..'*. 

3.3.2 Subcutaneous Route 

Significant responses were seen with codeine, but not with codeine 6- 
glucuronide or the intermediate after subcutaneous administration. There was 
also a difference in the peak response time. While codeine produced a peak 
response 30 minutes after administration, codeine 6-glucuronide and the 
intermediate showed a peak response 45 minutes after they were administered. 
The response time (Tables 3-17 to 3-20) for all the treatments was transformed 
into% of maximum effect data (Tables 3-21 to 3-24). The average values from 
the data sets were then plotted as a function of time (Figures 3-12 and 3-13). 
Codeine exhibited a significant response, that is, 61 ± 5% of the MPE (Figure 3- 



.i'N.'"-»-*i'7 '.•?'• 



^,*.f;lfJ">l'"'M"W, 



71 



13). On the other hand, codeine 6-glucuronide and the intermediate produced 
very poor responses, that is, 17 ± 3 and 1 1 + 2% of MPE, respectively. The% 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





10.7 


10.4 


10.4 


13.3 


11.9 


13.1 


11.6 


1.3 


0.5 


10 


28.1 


18.9 


18.8 


30.6 


26.3 


18.6 


23.6 


5.4 


2.2 


20 


33.3 


40.0 


38.1 


40.0 


38.9 


31.6 


37.0 


3.6 


1.5 


30 


29.2 


40.0 


33.3 


40.0 


37.2 


29.7 


34.9 


4.9 


2.0 


40 


24.8 


40.0 


28.3 


36.6 


31.8 


23.6 


30.9 


6.5 


2.7 


60 


22.6 


33.7 


24.2 


32.7 


27.7 


21.7 


27.1 


5.2 


2.1 


90 


15.2 


27.6 


22.7 


30.5 


26.9 


18.6 


23.6 


5.8 


2.4 


120 


13.3 


24.4 


20.9 


26.9 


24.8 


18.0 


21.4 


5.1 


2.1 


150 


13.4 


20.1 


19.7 


17.7 


19.9 


17.2 


18.0 


2.6 


1.0 


180 


12.1 


11.3 


16.8 


15.1 


12.6 


16.6 


14.1 


2.4 


1.0 



Table 3-5 : The data for the effect of intracerebroventricular 
administration of 10 ^g of codeine 6-glucuronide on the response time 
(in seconds). 



72 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





9.8 


9.4 


11.3 


8.3 


12.1 


9.4 


10.1 


1.4 


0.6 


10 


11.1 


16.3 


16.4 


15.8 


18.4 


16.6 


15.8 


2.5 


1.0 


20 


28.8 


27.6 


28.4 


29.7 


29.3 


29.7 


28.9 


0.8 


0.3 


30 


24.3 


25.9 


26.7 


27.8 


27.1 


26.8 


26.4 


1.2 


0.5 


40 


21.5 


22.7 


23.6 


24.4 


25.2 


24.5 


23.7 


1.4 


0.6 


60 


18.8 


21.1 


19.5 


21.1 


22.1 


23.7 


21.1 


1.8 


0.7 


90 


16.6 


19.8 


16.9 


17.9 


18.6 


20.9 


18.5 


1.7 


0.7 


120 


15.1 


16.5 


15.5 


14.2 


16.8 


19.6 


16.3 


1.9 


0.8 


150 


13.6 


11.4 


12.1 


11.7 


15.9 


16.2 


13.5 


2.1 


0.9 


180 


10.1 


9.2 


10.7 


8.4 


13.7 


11.3 


10.6 


1.9 


0.8 



Table 3-6 : The data for the effect of intracerebroventricular 
administration of 100 ng of codeine on the response time (in seconds). 



73 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





14.4 


11.4 


14.2 


10.0 


12.9 


12.7 


12.6 


1.7 


0.7 


10 


40.0 


14.6 


22.3 


33.1 


19.7 


22.6 


25.4 


9.4 


3.8 


20 


40.0 


28.1 


40.0 


40.0 


28.1 


32.8 


34.8 


5.9 


2.4 


30 


40.0 


24.4 


36.4 


40.0 


27.3 


30.7 


33.1 


6.7 


2.7 


40 


28.3 


19.3 


28.8 


40.0 


21.0 


24.6 


27.0 


7.4 


3.0 


60 


18.1 


19.8 


24.9 


40.0 


20.3 


20.4 


23.9 


8.2 


3.3 


90 


18.6 


15.3 


21.7 


29.3 


17.7 


17.8 


20.1 


5.0 


2.0 


120 


14.7 


11.1 


19.2 


27.8 


16.9 


16.3 


17.7 


5.6 


2.3 


150 


13.6 


10.7 


18.6 


22.7 


15.8 


14.7 


16.0 


4.2 


1.7 


180 


12.8 


9.8 


16.4 


14.6 


13.2 


12.3 


13.2 


2.2 


0.9 



Table 3-7 : The data for the effect of intracerebroventricular 
administration of 10 |ig of the intermediate on the response time 
(in seconds). 



74 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





11.2 


12.6 


12.3 


12.6 


8.7 


9.3 


11.1 


1.7 


0.7 


10 


30.2 


33.6 


24.2 


27.3 


26.9 


21.7 


27.3 


4.2 


1.7 


20 


40.0 


40.0 


40.0 


40.0 


40.0 


40.0 


40.0 


0.0 


0.0 


30 


40.0 


40.0 


40.0 


40.0 


40.0 


40.0 


40.0 


0.0 


0.0 


40 


40.0 


40.0 


40.0 


40.0 


40.0 


38.7 


39.8 


0.5 


0.2 


60 


33.6 


40.0 


40.0 


40.0 


40.0 


40.0 


38.9 


2.6 


1.1 


90 


24.7 


40.0 


40.0 


36.6 


34.2 


40.0 


35.9 


6.0 


2.4 


120 


18.9 


28.9 


26.7 


29.1 


27.7 


27.3 


26.4 


3.8 


1.6 


150 


18.6 


19.7 


20.1 


20.6 


19.9 


22.1 


20.2 


1.2 


0.5 


180 


13.8 


16.6 


14.3 


14.7 


12.3 


13.4 


14.2 


1.4 


0.6 



Table 3-8 : The data for the effect of intracerebroventricular 
administration of 5 i^g of morphine on the response time (in seconds). 



75 



===== 
Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





11.2 


10.4 


10.3 


11.0 


11.4 


8.7 


10.5 


1.0 


0.4 


10 


10.6 


11.7 


11.2 


12.8 


13.1 


9.9 


11.6 


1.2 


0.5 


20 


12.1 


12.3 


12.3 


13.2 


13.6 


10.2 


12.3 


1.2 


0.5 


30 


14.4 


11.6 


14.3 


12.6 


12.9 


10.9 


12.8 


1.4 


0.6 


40 


15.2 


10.9 


14.8 


13.9 


13.9 


11.3 


13.3 


1.8 


0.7 


60 


14.1 


11.1 


12.5 


14.1 


12.6 


10.6 


12.5 


1.5 


0.6 


90 


12.9 


10.8 


13.8 


12.7 


13.1 


9.7 


12.2 


1.6 


0.6 


120 


11.4 


11.2 


14.1 


13.6 


13.6 


11.3 


12.5 


1.4 


0.6 


150 


11.0 


11.6 


12.2 


12.1 


13.4 


11.5 


12.0 


0.8 


0.3 


180 


10.5 


10.5 


9.8 


11.2 


13.3 


10.8 


11.0 


1.2 


0.5 



Table 3-9 : The data for the effect of intracerebroventricular 
administration of saline on the response time (in seconds). 



76 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


4.3 


22.5 


17.8 


23.7 


22.6 


23.5 


19.1 


7.6 


3.1 


20 


62.9 


59.5 


59.6 


67.5 


61.6 


66.3 


62.9 


3.4 


1.4 


30 


48.0 


53.9 


53.7 


61.5 


53.8 


56.9 


54.6 


4.4 


1.8 


40 


38.7 


43.5 


42.9 


50.8 


47.0 


49.3 


45.4 


4.5 


1.8 


60 


29.8 


38.2 


28.6 


40.4 


35.8 


46.7 


36.6 


6.8 


2.8 


90 


22.5 


34.0 


19.5 


30.3 


23.3 


37.6 


27.9 


7.2 


2.9 


120 


17.5 


23.2 


14.6 


18.6 


16.8 


33.3 


20.7 


6.8 


2.8 


150 


12.6 


6.5 


2.8 


10.7 


13.6 


22.2 


11.4 


6.7 


2.7 


180 


1.0 


-0.7 


-2.1 


0.3 


5.7 


6.2 


1.8 


3.4 


1.4 



Table 3-10 : The data for the effect of intracerebroventricular 
administration of 100 jig of codeine on the analgesic responses 
(in%MPE). 



77 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


59.4 


28.7 


28.4 


64.8 


51.2 


20.4 


42.2 


18.6 


7.6 


20 


77.1 


100.0 


93.6 


100.0 


96.1 


68.8 


89.3 


13.1 


5.4 


30 


63.1 


100.0 


77.4 


100.0 


90.0 


61.7 


82.0 


17.3 


7.1 


40 


48.1 


100.0 


60.5 


87.3 


70.8 


39.0 


67.6 


23.2 


9.5 


60 


40.6 


78.7 


46.6 


72.7 


56.2 


32.0 


54.5 


18.3 


7.5 


90 


15.4 


58.1 


41.6 


64.4 


53.4 


20.4 


42.2 


20.3 


8.3 


120 


8.9 


47.3 


35.5 


50.9 


45.9 


18.2 


34.5 


17.2 


7.0 


150 


9.2 


32.8 


31.4 


16.5 


28.5 


15.2 


22.3 


9.9 


4.0 


180 


4.8 


3.0 


21.6 


6.7 


2.5 


13.0 


8.6 


7.4 


3.0 



Table 3-1 1 : The data for the effect of intracerebroventricular 
administration of 10 |ag of codeine 6-glucuronide on the analgesic 
responses (in% MPE). 



78 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


66.0 


76.6 


43.0 


53.6 


58.1 


40.4 


56.3 


13.8 


5.6 


20 


100.0 


100.0 


100.0 


100.0 


100.0 


100.0 


100.0 


0.0 


0.0 


30 


100.0 


100.0 


100.0 


100.0 


100.0 


100.0 


100.0 


0.0 


0.0 


40 


100.0 


100.0 


100.0 


100.0 


100.0 


95.8 


99.3 


1.7 


0.7 


60 


77.8 


100.0 


100.0 


100.0 


100.0 


100.0 


96.3 


9.1 


37 


90 


46.9 


100.0 


100.0 


87.6 


81.5 


100.0 


86.0 


20.7 


8.4 


120 


26.7 


59.5 


52.0 


60.2 


60.7 


58.6 


53.0 


13.2 


5.4 


150 


25.7 


25.9 


28.2 


29.2 


35.8 


41.7 


31.1 


6.4 


2.6 


180 


9.0 


14.6 


7.2 


7.7 


11.5 


13.4 


10.6 


3.1 


1.3 



Table 3-12 : The data for the effect of intracerebroventricular 
administration of 5 jig of morphine on the analgesic responses 
(in% MPE). 



79 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


100.0 


11.2 


31.4 


77.0 


25.1 


36.3 


46.8 


34.1 


13.9 


20 


100.0 


58.4 


100.0 


100.0 


56.1 


73.6 


81.4 


21.3 


8.7 


30 


100.0 


45.5 


86.0 


100.0 


53.1 


65.9 


75.1 


23.7 


9.7 


40 


54.3 


27.6 


56.6 


100.0 


29.9 


43.6 


52.0 


26.4 


10.8 


60 


14.5 


29.4 


41.5 


100.0 


27.3 


28.2 


40.1 


30.6 


12.5 


90 


16.4 


13.6 


29.1 


64.3 


17.7 


18.7 


26.6 


19.2 


7.8 


120 


1.2 


-1.0 


19.4 


59.3 


14.8 


13.2 


17.8 


21.9 


8.9 


150 


-3.1 


-2.4 


17.1 


42.3 


10.7 


7.3 


12.0 


16.8 


6.8 


180 


-6.3 


-5.6 


8.5 


15.3 


1.1 


-1.5 


1.9 


8.5 


3.5 



Table 3-13 : The data for the effect of intracerebroventricular 
administration of the 10 ng of the intermediate on the analgesic 
responses (in% MPE). 



Sv* ■■< 



t v-y-*" - ■ ■^■<r-^ -^.r* -F^' 



80 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


-2.1 


4.4 


3.0 


6.2 


5.9 


3.8 


3.6 


3.0 


1.2 


20 


3.1 


6.4 


6.7 


7.6 


7.7 


4.8 


6.1 


1.8 


0.7 


30 


11.1 


4.1 


13.5 


5.5 


5.2 


7.0 


7.7 


3.7 


1.5 


40 


13.9 


1.7 


15.2 


10.0 


8.7 


8.3 


9.6 


4.8 


2.0 


60 


10.1 


2.4 


7.4 


10.7 


4.2 


6.1 


6.8 


3.3 


1.3 


90 


5.9 


1.4 


11.8 


5.9 


5.9 


3.2 


5.7 


3.5 


1.4 


120 


0.7 


2.7 


12.8 


9.0 


7.7 


8.3 


6.9 


4.4 


1.8 


150 


-0.7 


4.1 


6.4 


3.8 


7.0 


8.9 


4.9 


3.4 


1.4 


180 


-2.4 


0.3 


-1.7 


0.7 


6.6 


6.7 


1.7 


4.0 


1.6 



Table 3-14 : The data for the effect of intracerebroventricular 
administration of saline on the analgesic responses (in% MPE). 



81 



40^ 





30 


o 




o 




« 








o 




E 


20 


h- 




m 

Q. 




0) 




O 




0^ 


10 




■ ■ I ■ 

30 



+-f 



+ 



60 



90 



-.— I— 

120 150 



Time (min) 



Cod 

(100 meg) 
.C6G 
(10 meg) 
Mor 
(5 meg) 
Int 

(10 meg) 
Saline 



180 



* (meg = micrograms). .V' -, 

Figure 3-9 : Effect of Response Time to ICV Administration 

of Saline, Codeine, Intermediate, Codeine 6-Giucuronide and Morphine. 



Compound 


% MPE 


Peak Time 


Morphine (5 |ig) 


100 ±- 


20 min 


Codeine (100 |ig) 


63 + 2 


20 min 


Codeine 6-glucuronide (10 [ig) 


89 ± 6 


20 min 


Intermediate (10 i^g) 


81 ± 9 


20 min 



Table 31-5 : % of maximum possible effect for various compounds at 
the peak response time after i.c.v. administration. 



82 



o 

£ 

UJ 

E 

3 

E 

X 

(0 




C6G 

(10 meg) 
Int 
(10 meg) 

Cod 

(100 meg) 
Saline 

Mor 
(5 meg) 



30 60 90 120 150 180 
Time (min) 



* (meg = micrograms). 

Figure 3-10 : Analgesic responses of rats to i.c.v. administration 

of saline, codeine, intermediate, codeine 6-glucuronide and morphine. 



Compound 


Total AUECo.3h 


Morphine (5 ^ig) 


11 545 ±744 


Codeine (100 i^g) 


4462 ± 546 


Codeine 6-glucuronide (10 i^g) 


8156 ±1744 


Intermediate (10 ^g) 


6416 ±2602 



Table 3-16 : Total AUECo.3h values after i.c.v. administration. 



83 




C6G Sal Cod Sal Int Sal Mor Sal 



Figure 3-1 1 : The total area under the effect curve (AUEC0.3 h) after 
i.c.v. administration. 



The area under the effect curve (AUEC) was determined from individual% IVIPE 
versus time graphs for each treatment using trapezoidal calculation (Figure 3-14) 
and is summarized in Table 3-26. 

3.3.3 Intravenous Route 



Intravenous administration resulted in significant increases in the 
responses associated with both codeine 6-glucuronide and the intermediate. 
Codeine also produced a greater response by this route compared to the 
subcutaneous route. As in the case of subcutaneous administration, there were 
differences in the peak response times. 



84 



Time (min] 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





10.9 


9.9 


11.6 


8.7 


10.8 


12.2 


10.7 


1.2 


0.5 


15 


18.2 


16.3 


17.4 


18.3 


20.5 


22.3 


18.8 


2.2 


0.9 


30 


26.9 


27.7 


28.9 


27.3 


29.9 


30.2 


28.5 


1.4 


0.6 


45 


21.3 


25.1 


26.3 


26.6 


28.7 


28.6 


26.1 


2.7 


1.1 


60 


20.6 


24.4 


24.7 


25.9 


28.3 


27.8 


25.3 


2.8 


1.1 


90 


18.7 


22.2 


23.6 


25.4 


27.9 


27.7 


24.3 


3.5 


1.4 


120 


17.6 


20.1 


21.1 


23.5 


25.8 


25.4 


22.3 


3.2 


1.3 


180 


16.8 


18.7 


20.3 


21.7 


22.6 


23.3 


20.6 


2.5 


1.0 


240 


14.9 


16.6 


17.5 


20.2 


20.6 


21.1 


18.5 


2.5 


1.0 


300 


13.8 


14.8 


15.2 


18.3 


17.7 


18.4 


16.4 


2.0 


0.8 


360 


12.3 


12.2 


13.7 


15.1 


13.2 


13.8 


13.4 


1.1 


0.4 



Table 3-17 : The data for the effect of subcutaneous administration of 
10 mg/kg of codeine on the response time (in seconds). 



,--». -T y, 1-^ f-j-T'^'^'T"' 1 "? r"- ;•> " ■•» 



85 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





8.8 


10.6 


10.3 


9.4 


13.1 


13.2 


10.6 


1.8 


0.6 


15 


12.3 


12.1 


12.5 


12.2 


11.3 


10.9 


11.9 


1.6 


0.5 


30 


14.7 


14.8 


12.8 


13.6 


12.1 


12.2 


13.5 


2.3 


0.8 


45 


18.2 


17.3 


14.7 


13.9 


12.7 


13.7 


15.3 


3.6 


1.2 


60 


16.8 


16.5 


12.9 


13.7 


14.6 


13.9 


14.9 


2.9 


1.0 


90 


16,7 


14.9 


12.6 


12.9 


13.3 


12.6 


13.9 


2.9 


1.0 


120 


13.0 


14.5 


13.5 


12.7 


13.1 


12.2 


13.2 


1.8 


0.6 


180 


11.4 


13.7 


11.7 


11.2 


12.7 


12.3 


11.8 


1.6 


0.5 


240 


9.8 


12.1 


11.2 


11.9 


11.9 


11.9 


11.3 


1.2 


0.4 


300 


10.4 


11.3 


11.8 


10.8 


11.3 


11.8 


11.3 


0.7 


0.2 


360 


8.9 


11.0 


11.9 


10.3 


10.8 


11.2 


10.6 


1.1 


0.4 



Table 3-18 : The data for the effect of subcutaneous administration of 
10 mg/kg of codeine 6-glucuronide on the response time (in seconds). 



Time (min] 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





9.5 


9.9 


10.1 


10.2 


12.1 


10.1 


10.0 


1.0 


0.3 


15 


9.8 


11.5 


10.6 


10.4 


11.3 


9.5 


10.3 


0.8 


0.3 


30 


10.2 


13.3 


10.6 


12.3 


10.9 


11.2 


11.3 


1.1 


0.4 


45 


12.5 


13.8 


11.3 


13.8 


12.6 


11.6 


13.0 


1.3 


0.5 


60 


13.2 


13.7 


12.5 


13.9 


13.1 


12.8 


13.2 


0.7 


0.2 


90 


13.4 


12.6 


12.9 


12.9 


13.3 


12.7 


13.0 


1.2 


0.4 


120 


12.7 


12.7 


11.7 


12.6 


12.8 


11.9 


12.6 


1.1 




0.4 


180 


11.9 


13.2 


12.3 


11.8 


12.2 


11.3 


12.1 


0.6 


0.2 


240 


12.6 


12.5 


12.6 


11.9 


11.7 


10.8 


11.9 


1.1 


0.4 


300 


12.7 


11.9 


10.7 


11.1 


11.6 


10.7 


11.4 


0.8 


0.3 


360 


12.0 


10.7 


9.6 


11.4 


10.3 


10.2 


10.8 


1.0 


0.3 



Table 3-19 : The data for the effect of subcutaneous administration 
of 10 mg/kg of the intermediate on the response time (in seconds). 



87 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





8.1 


8.5 


8.6 


9.8 


9.6 


10.8 


9.8 


1.4 


0.5 


15 


9.8 


10.0 


8.1 


10.7 


9.3 


9.2 


9.8 


0.9 


0.3 


30 


10.1 


10.8 


10.2 


10.2 


10.0 


9.9 


10.3 


0.5 


0.2 


45 


11.0 


10.2 


11.3 


10.1 


10.1 


11.2 


10.9 


0.7 


0.3 


60 


9.8 


11.1 


9.8 


10.0 


9.9 


11.7 


10.6 


0.8 


0.3 


90 


8.9 


10.8 


9.8 


10.2 


10.2 


12.2 


10.6 


1.0 


0.4 


120 


8.6 


9.3 


10.7 


10.3 


10.3 


11.9 


10.3 


1.0 


0.4 


180 


10.3 


8.9 


11.3 


9.5 


10.7 


11.7 


10.5 


0.9 


0.3 


240 


10.6 


9.4 


10.7 


9.7 


9.8 


10.6 


10.1 


0.5 


0.2 


300 


11.2 


8.6 


11.3 


9.9 


9.9 


10.4 


9.9 


1.0 


0.4 


360 


11.1 


9.7 


10.6 


10.1 


10.4 


10.2 


10.2 


0.6 


0.2 



Table 3-20 : The data for the effect of subcutaneous administration 
of saline on the response time (in seconds). 



> . f 



88 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


15 


25.1 


21.3 


20.4 


30.7 


33.2 


36.3 


27.8 


6.6 


2.7 


30 


55.0 


59.1 


60.9 


59.4 


65.4 


64.7 


60.8 


3.9 


1.6 


45 


35.7 


50.5 


51.8 


57.2 


61.3 


59.0 


52.6 


9.2 


3.8 


60 


33.3 


48.2 


46.1 


55.0 


59.9 


56.1 


49.8 


9.6 


3.9 


90 


26.8 


40.9 


42.3 


53.4 


58.6 


55.8 


46.3 


12.0 


4.9 


120 


23.0 


33.9 


33.5 


47.3 


51.4 


47.5 


39.4 


11.0 


4.5 


180 


20.3 


29.2 


30.6 


41.5 


40.4 


39.9 


33.7 


8.4 


3.4 


240 


13.7 


22.3 


20.8 


36.7 


33.6 


32.0 


26.5 


8.9 


3.6 


300 


10.0 


16.3 


12.7 


30.7 


23.6 


22.3 


19.3 


7.7 


3.1 


360 


4.8 


7.6 


7.4 


20.4 


8.2 


5.8 


9.0 


5.7 


2.3 



Table 3-21 : The data for the effect of subcutaneous administration of 
10 mg/kg of codeine on the analgesic responses (in% MPE). 



89 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


15 


4.5 


5.1 


10.2 


7.4 


2.8 


9.2 


6.9 


3.0 


1.0 


30 


11.6 


14.3 


21.6 


8.4 


5.9 


13.7 


12.8 


6.1 


2.0 


45 


28.4 


22.8 


28.3 


14.8 


2.5 


14.7 


16.6 


9.8 


3.3 


60 


27.1 


20.1 


21.9 


8.8 


4.7 


14.1 


13.9 


7.3 


2.4 


90 


18.8 


14.6 


21.2 


7.7 


0.6 


11.4 


11.1 


7.7 


2.6 


120 


9.6 


13.3 


16.3 


10.8 


5.6 


10.8 


11.3 


3.9 


1.3 


180 


4.1 


10.5 


5.7 


4.7 


1.6 


5.9 


5.7 


3.2 


1.1 


240 


0.0 


5.1 


3.5 


3.0 


3.7 


8.2 


4.7 


2.1 


0.7 


300 


1.0 


2.4 


2.8 


5.1 


8.1 


4.6 


4.6 


2.3 


0.8 


360 


2.7 


1.4 


-3.5 


5.4 


3.1 


2.9 


1.9 


3.3 


'' 



Table 3-22 : The data for the effect of subcutaneous administration of 
10 mg/kg of the Intermediate on the analgesic responses (in% MPE). 



'^n •i^-' - --^r^- 



. -, *-■• 



90 



Time (min; 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


15 


0.3 


1.0 


5.3 


1.7 


3.8 


0.7 


1.6 


2.8 


1.0 


30 


3.3 


2.3 


11.3 


1.7 


8.9 


7.0 


4.5 


5.7 


2.0 


45 


19.0 


9.8 


13.0 


4.0 


14.7 


12.1 


11.1 


4.2 


1.5 


60 


15.7 


12.1 


12.6 


8.0 


11.2 


12.4 


10.0 


3.6 


1.3 


90 


18.6 


12.8 


9.0 


9.4 


7.0 


9.1 


8.6 


2.8 


1.0 


120 


18.0 


10.5 


9.3 


5.4 


7.7 


8.1 


7.2 


2.9 


1.0 


180 


10.5 


7.9 


11.0 


7.4 


9.3 


5.4 


6.9 


3.7 


1.3 


240 


11.4 


10.2 


8.6 


8.4 


3.5 


5.7 


5.8 


4.3 


1.5 


300 


8.8 


10.5 


6.6 


2.0 


5.1 


3.0 


4.2 


4.2 


1.5 


360 

1 


9.5 


8.2 


2.7 


-1.7 


4.8 


4.0 


1.9 


5.2 


1.8 



Table 3-23 : The data for the effect of subcutaneous administration 
of 10 mg/kg of codeine 6-glucuronide on the analgesic responses 
(in% MPE). 



91 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


15 


11.3 


11.6 


13.1 


13.9 


8.3 


0.7 


8.0 


6.5 


2.5 


30 


15.6 


17.5 


16.9 


20.8 


16.2 


-1.1 


11.8 


9.7 


3.7 


45 


-2.8 


13.4 


2.1 


20.2 


17.8 


-7.1 


6.4 


10.7 


4.0 


60 


-1.1 


14.4 


7.6 


21.5 


15.0 


-4.9 


7.7 


9.7 


3.7 


90 


-4.3 


9.1 


3.8 


18.6 


12.7 


-0.4 


5.5 


8.3 


3.1 


120 


-1.1 


7.2 


10.3 


17.0 


13.1 


-2.8 


6.2 


7.7 


2.9 


180 


-5.0 


11.6 


-3.1 


17.7 


12.4 


-1.8 


4.7 


9.0 


3.4 


240 


-1.4 


10.0 


5.5 


15.8 


10.5 


-3.2 


5.1 


7.4 


2.8 


300 


-6.7 


5.9 


2.1 


18.3 


10.5 


-7.4 


2.8 


9.5 


3.6 


360 


-5.0 


3.8 


-7.9 


12.6 


9.9 


-1.8 


1.0 


7.9 


3.0 



Table 3-24 : The data for the effect of subcutaneous administration 
of saline on the analgesic responses (in% MPE). 



92 



o 

o 

E 

H 



CO 

o 



Saline 

Int 

10 mg/k 

C6G 
10 mg/k 

Codeine 
10 mg/k 




I I 1 1 1 1 I I 1 1 1 1 1 1 

180 240 300 360 



Time (min) 



Figure 3-12 : Effect of response time to s.q. administration 
of saline, codeine, intermediate and codeine 6-glucuronide. 



Compound 


% MPE 


Peak Time 


Codeine (10 mg/kg) 


61 ± 5 


30 min 


Codeine 6-glucuronide ( 10 mg/kg) 


11 ± 2 


45 min 


Intermediate (10 mg/kg) 


17 ± 3 


45 min 11 



Tgble 3-25 : % of maximum possible effect for various compounds at 
the peak response time after s.q. administration. 



93 



o 
UJ 

E 

3 

E 

X 



S5 




60 120 180 240 

Time (min) 



300 



360 



Figure 3-13 ; Analgesic responses to s.q. administration 
of saline, codeine, intermediate and codeine 6-glucuronide. 



1 Compound 


Total AUECo.6h 


Codeine (10 mg/kg) 


10886 ± 646 


Codeine 6-glucuronide (10 mg/kg) 


1765 ± 159 


Intermediate (10 mg/kg) 


2321 ± 269 



Table 3-26 : Total AUECo.6 h values after s.q. administration. 



94 



15000 ^ 



12000 . . 



2 9000 
Ul 

< 

CO 6000 

o 



3000 . . 



ii 



f 



U 



C6G Sal Cod Sal Int 



Sal 



Figure 3-14 : The total area under the effect curve (AUECo^h) after 
s.q. administration. 



The response time (Tables 3-27 to 3-30) for all the treatments was transformed 
into% of maximum effect data (Tables 3-31 to 3-34). The average values from 
the data sets were then plotted as a function of time (Figures 3-15 and 3-16). 
Codeine exhibited a significant response, that is, 98 ± 4% of the MPE. Codeine 
6-glucuronide and the intermediate produced responses which were 55 + 3 and 
66 ± 3% of MPE, respectively. The% MPE at peak response time for all the 
compounds is summarized in Table 3-35. The area under the effect curve 
(AUEC) was determined from individual% MPE versus time graphs for each 
treatment using trapezoidal calculation (Figure 3-17) and is summarized in Table 
3-36. 



95 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





8.7 


11.8 


9.8 


12.8 


10.1 


11.4 


11.1 


1.6 


0.6 


10 


27.6 


31.3 


33.7 


31.7 


29.8 


27.7 


29.3 


3.4 


1.3 


20 


38.4 


37.9 


36.9 


40.0 


39.1 


37.2 


38.5 


1.2 


0.5 


30 


35.9 


34.1 


33.6 


37.7 


36.3 


34.5 


35.7 


3.1 


1.2 


40 


32.7 


33.8 


32.9 


34.3 


35.4 


32.8 


33.3 


3.6 


1.4 


60 


23.8 


22.2 


26.9 


26.4 


30.6 


25.1 


25.7 


2.7 


1.0 


90 


19.7 


18.1 


22.7 


23.1 


24.3 


23.7 


21.7 


2.3 


0.9 


120 


18.1 


17.7 


20.3 


20.3 


21.0 


18.8 


19.0 


1.6 


0.6 


180 


14.8 


15.2 


19.7 


19.7 


19.9 


16.3 


17.2 


2.5 


0.9 


240 


13.2 


14.4 


16.8 


16.6 


9.4 


13.2 


13.8 


2.5 


0.9 


300 


11.6 


12.3 


14.7 


14.5 


10.0 


12.7 


12.6 


1.6 


0.6 



Table 3-27 : The data for the effect of intravenous administration of 
10 mg/kg of codeine on the response time (in seconds). 



96 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





12.1 


11.7 


10.6 


12.6 


10.3 


11.6 


11.7 


0.9 


0.4 


10 


19.9 


17 


19.7 


21.2 


14.4 


13.9 


17.0 


3.3 


1.3 


20 


26.6 


25.8 


26.9 


25.7 


23.9 


22.1 


24.5 


2.5 


0.9 


30 


28.2 


29.6 


28.3 


29.5 


26.4 


25.3 


27.3 


2.2 


0.8 


40 


26.7 


27.7 


26.2 


22.3 


24.1 


23.2 


24.5 


2.5 


0.9 


60 


19.4 


22.3 


24.7 


19.9 


22.9 


20.8 


20.9 


2.7 


1.0 


90 


16.3 


18.8 


22.3 


17.8 


17.7 


17.8 


18.1 


2.1 


0.8 


120 


14.7 


16.9 


20.9 


15.6 


15.5 


16.9 


16.4 


2.2 


0.8 


180 


13.9 


14.6 


19.2 


14.4 


14.1 


15.4 


15.1 


1.9 


0.7 


240 


12.6 


11.8 


18.1 


13.3 


12.8 


14.8 


13.8 


2.1 


0.8 


300 


11.8 


10.9 


16.3 


12.8 


10.9 


13.9 


12.8 


1.9 


0.7 



Table 3-28 : The data for the effect of intravenous administration of 
10 mg/kg of codeine 6-glucuronide on the response time (in seconds). 



97 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





10.1 


11.6 


9.1 


12.2 


11.2 


13 


11.3 


1.3 


0.5 


10 


17.6 


20.5 


15.7 


19.1 


17.5 


18.2 


17.9 


1.6 


0.6 


20 


23.9 


24.8 


24.2 


24.5 


23.6 


25.1 


24.4 


0.5 


0.2 


30 


32.7 


33.9 


30.7 


29.1 


28.7 


29.6 


30.4 


2.2 


0.8 


40 


27.9 


26.5 


25.1 


25.2 


26.0 


27.7 


25.8 


2.0 


0.8 


60 


25.4 


19.0 


23.2 


23.9 


22.3 


22.3 


22.4 


2.1 


0.8 


90 


22.4 


17.6 


18.1 


21.2 


20.8 


21.1 


20.0 


1.8 


0.7 


120 


18.8 


16.5 


16.4 


18.4 


17.7 


19.7 


18.0 


1.2 


0.5 


180 


14 


15.6 


14.8 


17.1 


17.3 


17.8 


16.3 


1.5 


0.6 


240 


13.7 


14.8 


12.7 


16.2 


16.6 


16.9 


15.1 


1.6 


0.6 


300 


12.8 


13.1 


11.1 


15.5 


14.7 


14.6 


13.5 


1.5 


0.6 



Table 3-29 : The data for the effect of intravenous administration of 
10 mg/kg of the intermediate on the response time (in seconds). 



1 • « 'f i 



98 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





9.3 


9.9 


11.1 


10.1 


13.0 


12.2 


11.2 


1.5 


0.6 


10 


10.8 


10.3 


10.7 


10.4 


12.7 


12.8 


11.5 


1.2 


0.5 


20 


10.6 


10.6 


11.0 


11.1 


11.8 


13.1 


11.6 


1.0 


0.4 


30 


11.3 


10.8 


11.9 


10.8 


13.5 


12.7 


12.0 


1.1 


0.4 


40 


11.0 


11.1 


12.3 


10.7 


12.2 


12.2 


11.9 


1.0 


0.4 


60 


10.9 


11.2 


11.6 


11.1 


13.3 


12.8 


12.1 


1.2 


0.5 


90 


11.3 


11.2 


11.7 


10.0 


12.7 


12.9 


11.8 


1.1 


0.4 


120 


11.6 


10.8 


12.2 


11.5 


12.2 


12.3 


11.9 


0.7 


0.3 


180 


10.9 


10.9 


11.9 


11.3 


13 


12.6 


11.9 


0.9 


0.3 


240 


11.1 


10.6 


11.3 


10.6 


13.1 


13.6 


11.9 


1.3 


0.5 


300 


10.6 


10.9 


11.9 


9.8 


13.2 


13.2 


11.7 


1.3 


0.5 



Table 3-30 : The data for the effect of intravenous administration of 
saline on the response time (in seconds). 



99 



Time (min) 


1 


2 


3 


4 


5 


6 


MEAN 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


60.4 


69.1 


79.1 


69.5 


65.9 


57.0 


66.8 


7.1 


2.9 


20 


97.9 


99.6 


89.7 


100.0 


97.0 


94.2 


98.3 


13.3 


4.2 


30 


86.9 


79.1 


78.8 


91.5 


87.6 


80.8 


84.1 


4.8 


2.0 


40 


76.7 


78.0 


76.5 


79.0 


84.6 


74.8 


78.3 


3.1 


1.3 


60 


48.2 


36.9 


56.6 


50.0 


68.6 


47.9 


51.4 


9.6 


3.9 


90 


35.1 


22.3 


42.7 


37.9 


47.5 


43.0 


36.6 


9.0 


3.4 


120 


30.0 


20.9 


34.8 


27.6 


36.5 


25.9 


27.1 


7.7 


2.9 


180 


19.5 


12.1 


32.8 


25.4 


32.8 


17.1 


20.9 


10.0 


3.8 


240 


14.4 


9.2 


23.2 


14.0 


-2.3 


6.3 


9.5 


8.6 


3.3 


300 


9.3 


1.8 


16.2 


6.3 


-0.3 


4.5 


5.0 


6.4 


2.4 



Table 3-31 : The data for the effect of intravenous administration of 
10 mg/kg of codeine on the analgesic responses (in% MPE). 



'(. »j/ - ' - -. >, -I .' f » 1 



100 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


28.0 


18.7 


31.0 


31.4 


13.8 


8.1 


18.8 


12.0 


4.5 


20 


52.0 


49.8 


55.4 


47.8 


45.8 


37.0 


45.0 


9.8 


3.7 


30 


57.7 


63.3 


60.2 


61.7 


54.2 


48.2 


55.2 


8.1 


3.1 


40 


52.3 


56.5 


53.1 


35.4 


46.5 


40.8 


45.0 


9.8 


3.7 


60 


26.2 


37.5 


48.0 


26.6 


42.4 


32.4 


32.4 


11.4 


4.3 


90 


15.1 


25.1 


39.8 


19.0 


24.9 


21.8 


22.4 


9.2 


3.5 


120 


9.3 


18.4 


35.0 


10.9 


17.5 


18.7 


16.5 


9.6 


3.6 


180 


6.5 


10.2 


29.3 


6.6 


12.8 


13.4 


11.8 


8.4 


3.2 


240 


1.8 


0.4 


25.5 


2.6 


8.4 


11.3 


7.4 


8.9 


3.4 


300 


-1.1 


-2.8 


19.4 


0.7 


2.0 


8.1 


3.9 


7.6 


2.9 



Table 3-32 : The data for the effect of intravenous administration of 
10 mg/l<g of codeine 6-glucuronide on the analgesic responses 
(in% MPE). 



101 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


25.1 


31.3 


21.4 


24.8 


21.9 


19.3 


22.8 


4.9 


1.8 


20 


46.2 


46.5 


48.9 


44.2 


43.1 


44.8 


45.5 


1.9 


0.7 


30 


75.6 


78.5 


69.9 


60.8 


60.8 


61.5 


66.2 


8.5 


3.2 


40 


59.5 


52.5 


51.8 


46.8 


51.4 


54.4 


50.2 


7.7 


2.9 


60 


51.2 


26.1 


45.6 


42.1 


38.5 


34.4 


38.3 


8.8 


3.3 


90 


41.1 


21.1 


29.1 


32.4 


33.3 


30.0 


30.3 


6.4 


2.4 


120 


29.1 


17.3 


23.6 


22.3 


22.6 


24.8 


23.1 


3.5 


1.3 


180 


13.0 


14.1 


18.4 


17.6 


21.2 


17.8 


17.5 


3.0 


1.1 


240 


12.0 


11.3 


11.7 


14.4 


18.8 


14.4 


13.2 


3.0 


1.1 


300 


9.0 


5.3 


6.5 


11.9 


12.2 


5.9 


7.6 


3.6 


1.3 



Table 3-33 : The data for the effect of intravenous administration of 
10 mg/kg of the intermediate on the analgesic responses (in % MPE). 



102 



Time (min) 


1 


2 


3 


4 


5 


6 


Mean 


SD 


SEM 





0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


10 


4.9 


1.3 


-1.4 


1.0 


-1.1 


2.2 


0.9 


2.2 


0.8 


20 


4.2 


2.3 


-0.3 


3.3 


-4.4 


3.2 


1.0 


3.1 


1.2 


30 


6.5 


3.0 


2.8 


2.3 


1.9 


1.8 


2.8 


1.8 


0.7 


40 


5.5 


4.0 


4.2 


2.0 


-3.0 


0.0 


2.1 


2.9 


1.1 


60 


5.2 


4.3 


1.7 


3.3 


1.1 


2.2 


3.1 


1.5 


0.6 


90 


6.5 


4.3 


2.1 


-0.3 


-1.1 


2.5 


2.1 


2.7 


1.0 


120 


7.5 


3.0 


3.8 


4.7 


-3.0 


0.4 


2.3 


3.5 


1.3 


180 


5.2 


3.3 


2.8 


4.0 


0.0 


1.4 


2.2 


2.3 


0.9 


240 


5.9 


2.3 


0.7 


1.7 


0.4 


5.0 


2.2 


2.4 


0.9 


300 


4.2 


3.3 


2.8 


-1.0 


0.7 


3.6 


1.5 


2.8 


1.1 



Table 3-34 : The data for the effect of intravenous administration of 
saline on the analgesic responses (in% MPE). 



103 



u 

o 

E 

I- 

« 

a 

(0 

& 



Codeine 
10 mg/kg 

C6G 

10 mg/kg 

Int 

10 mg/kg 

Saline 




I I I » I I I I I I I I I I I ■ I I 

30 60 90 120 150 180 210 240 270 300 

Time (min) 



Figure 3-15 : Effect of response time to i.v. administration of saline, 
codeine, intermediate and codeine 6-glucuronide. 



1 Compound 


% MPE 


Peak Time 


Codeine (10 mg/kg) 


98 ± 4 


20 min 


Codeine 6-glucuronide ( 10 mg/kg) 


55 ± 3 


30 min 


Intermediate (10 mg/kg) 


66 ± 3 


30 min 



Table 3-35 : % of maximum possible effect for various compounds at 
the peak response time after i.v. administration. 



104 



o 







. 10 mg/kg 

a_C6G 
10 mg/kg 

^_Int 

10 mg/kg 
^_ Saline 



30 60 90 120 150 180 210 240 270 300 

Time (min) 



Figure 3-16 : Analgesic Responses to i.v. Administration of Saline, 
Codeine, Intermediate and Codeine 6-Glucuronide. 



1 Compound 


Total AUECo.5h 


Codeine (10 mg/kg) 


8718 ±960 


Codeine 6-glucuronide (10 mg/kg) 


4781 ± 589 


Intermediate (10 mg/kg) 


5933 ± 289 



Table 3-36 : Total AUECo-sh values after i.v. administration. 



105 



o 

O 
UJ 

3 
< 

(0 

o 



15000 ^ 



12000 . . 



9000 . . 



6000.. 



3000 . . 




C6G 



Sal 



Cod Sal 



Int Sal 



Figure 3-17 : The total area under the effect curve (AUEC0.5 n) after 
i.v. administration. 



3.4 Immune Studies 



Codeine and its 6-glucuronide demonstrated inhibition of proliferation in 
the PHA-stimulated lymphocytes, with maximal differences in cell inhibition of the 
parent compound and its metabolite in the lower, physiologically relevant 
concentration range of 0.156-5 jig/ml, as seen in Figure 3-18. Similarly, in the 
case of the PMA-stimulated lymphocytes, there was very little suppression 
observed at lower concentrations compared to the higher concentrations (Figure 
3-19). Codeine and codeine 6-glucuronide also inhibited the MLR-stimulated 
lymphocytes in a concentration-dependent fashion (Figure 3-20). 



106 




-20 



156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-18 : The % inhibition of proliferation of PHA-stimulated T 
lymphocytes after the addition of codeine and codeine 6-glucuronide. 
* and * * Indicate a p-value of 0.05 and 0.01 , respectively. 




313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-19 : The % inhibition of proliferation of PMA-stimulated T 
lymphocytes after the addition of codeine and codeine 6-glucuronide. 
* and * * indicate a p-value of 0.05 and 0.01 , respectively. 



107 



40 



gCod 
qC6G| 




156 



313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-20 : The% inhibition of proliferation of MLR-stimulated T 
lymphocytes after the addition of codeine and codeine 6-glucuronide. 



lymphocytes after the addition of codeine and codeine 6-glucuronide.Similar to 
the effects of codeine and codeine 6-glucuronide, morphine and its 6- 
glucuronide exhibited greatest inhibition of proliferation in the PHA-stimulated 
cells (Figure 3-21). In the case of PMA-stimulated lymphocytes (Figure 3-22), 
morphine and morphine 6-glucuronide demonstrated almost negligible inhibition 
at lower concentrations (<1.25 fig/ml). These compounds also showed Inhibition 
of the Mixed Lymphocyte Reaction (Figure 3-23). 

When the intermediate was tested against codeine 6-glucuronide, it 
showed a greater inhibition over the entire concentration range tested (0.156 to 
10 ^g/ml) in all of the assays, that is, PHA (Figure 3-24), PMA (Figure 3-25) and 
the MLR (Figure 3-26) models. 



108 



70 



60 

c 
o 

■^ 50 

I 
2 40 

Q. 
«*- 
O 

c 30 
o 

'■•3 

£ 20 



10.. 



|Mor 
0M6G 




156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-21 : The% inhibition of proliferation of PHA-stimulated T 
lymphocytes after the addition of morphine and morphine 6-glucuronide. 
* and * * indicate a p-value of 0.05 and 0.01 , respectively. 




156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-22 : The% inhibition of proliferation of PMA-stimulated T 
lymphocytes after the addition of morphine and morphine 6-glucuronide. 
* and * * indicate a p-value of 0.05 and 0.01, respectively. 



109 




156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-23 : The% inhibition of proliferation of MLR-stimulated T 
lymphocytes after the addition of morphine and morphine 6-glucuronide. 
* and * * indicate a p-value of 0.05 and 0.01 , respectively. 



60 



|lnt 
iC6G 




156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-24 : The% inhibition of proliferation of PHA-stimulated T 
lymphocytes after the addition of codeine 6-glucuronide and the 
intermediate. 



110 



c 
o 

♦3 

ig 

I 
o 

a. 

•3 

c 
o 

*3 

IS 

IE 
c 




156 313 625 1250 2500 5000 10000 
t Concentration (ng/ml) 



Figure 3-25 : The% inhibition of proliferation of PMA-stimulated T 
lymphocytes after the addition of codeine 6-glucuronide and the 
intermediate. 



40 



35. 



.2 30.. 
2 

(b 

S 25 4- 

o 

Q. 

•S 204- 

c 
o 

9 



15.. 



£ 10.. 
5i 



□ 



Int 
C8G 



Jl 




1-, 



156 313 625 1250 2500 5000 10000 
Concentration (ng/ml) 



Figure 3-26 : The% inhibition of proliferation of MLR-stimulated T 
lymphocytes after the addition of codeine 6-glucuronide and the 
intermediate. 



T T"^ IT," r ■« - 



111 



3.5 Receptor Binding Studies 

Morphine exhibited the highest affinity in the receptor binding assays, with 
an IC50 value of 1.8 ± 0.6 nM (Table 3-9). In the case of codeine, however, the 
binding curve was shifted significantly to the right of the concentration range 
(IC50 = 1.5 ± 0.4 |iM ) indicating a much weaker affinity for the |i receptor. 
Codeine 6-glucuronide had a slightly lower binding compared to codeine (IC50 = 
4.4 ± 0.2 fxM). The intermediate, on the other hand, exhibited a slightly higher ^ 
receptor binding affinity compared to codeine and codeine 6-glucuronide (IC50 = 
0.13 ± 0.06 ^M). 

The raw counts per minute (CPM) data of each compound were converted 
into the more standardized form, that is, % of total binding. The receptor binding 
profiles of morphine (Figures 3-27 and 3-28), codeine (Figures 3-29 and 3-30), 
codeine 6-glucuronide (Figures 3-31 and 3-32) and the intermediate (Figures 3- 
33 and 3-34) are represented in the graphs below. 



Parameters 


Mor 


Cod 


C6G 


Int 


T (cpm) 


3358 
±343 


2609 
±350 


2249 
±107 


2479 
±481 


NS (cpm) 


961 

±97 


876 
±24 


706 
±64 


779 
±141 


N 


0.98 
±0.42 


0.96 
±0.26 


1.02 
±0.28 


1.13 
±0.36 


IC50 


1.81 

±0.58 

nM 


1.49 
±0.39 

^M 


4.4 

±0.23 

HM 


0.13 

±0.06 

l^M 



Table 3-37 : The |i receptor binding parameters for morphine, codeine, 
codeine 6-glucuronide and the intermediate. 



112 






3.500 



2.900 



2.300 



1.700 - 



1.100 



0.500 

10-12 10" 10-i« 10-» 10-* 10-' 10-* 

Concentration (M) 

Figure 3-27 : The receptor binding profile of morphine. 
(CPM vs concentration). 




105 



1 

S 

1 




' ' ' I '— ^-n ' — ' ' I ' — n "— "-^ 

10-12 -I0-" 10" 10-» 10* 10-' io-» 

Concentration(M) 

Figure 3-28 : The receptor binding profile of morphine. 
(% total binding vs concentration). 



flL 

u 



2.600 



2.267 



1.933 



? 1.600 



1.267 



0.933 - 



0.600 



10' 



113 



Concentration (M) 



■^^T 1 TT-i 1 r-T^ 1 ^f] 1 — n-1 , r , 

10-* 10-' 10-* 10* 10-* 10-3 



Figure 3-29 : The receptor binding profile of codeine. 
(CPM vs concentration). 



1 
S 

I 




10-« 10-* 



10-7 10-« 10-5 

Concentration(M) 



10^ 



10- 



Figure 3-30 : The receptor binding profile of codeine. 
(% total binding vs concentration). 



114 



u 



z.u - 


*-. 


S^ 








1.8- 




X 








1.6- 


• " ■ 




\ 






14- 






\ 


r *^ 




1.2- 


■ 5 - 


■■'■: : 


...3 




'I ^ 


1.0- 




y 


;A /■■ 


\(''.' r. ' 




. 


■' "■- 


'.,■ 








0.8- 






' ' 'I' 


1 n <— 


— • 


0.6- 


• 

i-r-| ,—r-r 



10-9 10-« 10' io-« 10-* 

Concentration (M) 



io-» 



10-s 



Figure 3-31 : The receptor binding profile of codeine 6- 
glucuronide. (CPI\/1 vs concentration). 



1 



I 




10^ 10-* 10-' 1G-« 10* 

Concent ration(M) 



10-* 10-3 



Figure 3-32 : The receptor binding profile of codeine 6- 
glucuronide. (% total binding vs concentration). 



115 



e 

8 

M 
S 

a. 
u 



2.500 



2.100 - 



1.700 



1.300 - 



0.900 



0.500 



10^ 10-» 



10' 

Concentration (M) 



-q ^^^T ^^-q , r- 

10-" 10* 10-* 10-3 



Figure 3-33 : The receptor binding profile of the intermediate. 
(CPM vs concentration). 



at 
e 

e 

s 

I 




io-« io-« 10-' io-« 10-5 

Concentration (IM) 



10-« 



10-3 



Figure 3-34 : The receptor binding profile of the intermediate. 
(% total binding vs concentration). 



116 



3.6 Plasma and Brain Concentrations 

System 2 was used to determine concentrations of codeine, morphine and 
their glucuronides in rat plasma and brain samples. Extractions were done using 
disposable Clean Screen® columns as in the case of blank human urine used to 
develop the HPLC method. However, for the rat plasma and brain samples, a 
10% ammonium hydroxide solution in methanol was required to obtain good 
recoveries of the polar glucuronide metabolites. The recoveries were better than 
80% for all the compounds. 

Intracerebroventricular administration of codeine (Figure 3-35) in rats 
resulted in the partial conversion of codeine to morphine in the brain (Table 3- 
38). However, no drug was detected in the plasma. When codeine 6-glucuronide 
was administered directly into the brain, a small amount of morphine 6- 
glucuronide was detected in the brain (Table 3-39) but no drug was detected in 
the plasma. 

After subcutaneous administration of codeine, it was seen that some 
codeine was converted into morphine and morphine 3-glucuronide in the plasma. 
Neither morphine 6-glucuronide or codeine 6-glucuronide were detected in the 
plasma. However, both morphine 6-glucuronide and codeine 6-glucuronide were 
present in small quantities in the brain of some of the rats. After subcutaneous 
administration of codeine 6-glucuronide, no drug or its metabolites was detected 
either in the plasma or the brain. Therefore, this route of administration was not 
pursued. 

After intravenous administration of codeine (Figure 3-36), some of the 
codeine was converted into morphine, morphine 3-glucuronide and codeine 6- 
glucuronide were found in the plasma (Table 3-40). In the brain, small amounts 



117 



of both morphine 6-glucuronicle and codeine 6-glucuronide were detected (Table 
3-41). Codeine 6-glucuronide administration resulted in the presence of only 
codeine 6-glucuronide in the plasma (Table 3-42). However, in the brain, small 
amounts of both codeine 6-glucuronide and morphine 6-glucuronide were 
detected (Table 3-43). The results obtained after administration of codeine and 
codeine 6-glucuronide by the various routes are summarized in Table 3-44. 



Sample 
number 


Codeine 

(ng/g) 


Morphine 
(ng/g) 


1 


9652 


1803 


2 


7965 


1046 


3 


8242 


1287 


4 


7191 


1311 


5 


6647 


1207 


6 


8018 


1379 


Mean ± SD 


7953 + 1026 


1339 + 254 



Table 3-38 : Concentrations in the rat brain after i.c.v. administration 
of 100 |ig codeine. 



. — 1 ^ 

Sample 

number 


C6G 
(ng/g) 


M6G 
(ng/g) 


1 


815 


63 


2 


744 


43 


3 


917 


57 


4 


1033 


78 


5 


668 


53 


6 


791 


69 


Mean + SD 


828 + 130 


61 +12 



Table 3-39 : Concentrations in the rat brain after i.c.v. administration of 
10 fig codeine 6-glucuronide. 



118 




A- 



12.00 i4!oo is.oo i»;od 2o!od ii'.oo ' zi'.to 'le'.oo jg 



Figure 3-35 : Chromatogram of a brain sample after administration of 100 
fig of codeine i.c.v. in the rat. 



0.0«- 














0.05- 




2 










0.04- 




A 










0.03- 














0.02- 




M 

S 


t»' ' '- 




, '■'"-- •■•'' 




O.Ol- 


J 


V. 


8 

U 








0.00- 




r — ^ ---^ 







12 


00 


; r— T : 

14.00 


18.00 U.OO 


20.00 


22.00 


24:00 '2« 



Figure 3-36 : Chromatogram of a plasma sample after administration 
of 10 mg/kg codeine i.v. in the rat. 



119 



Sample 
number 


Codeine 
(ng/ml) 


Morphine 
(ng/ml) 


C6G 
(ng/ml) 


M3G 
(ng/ml) 


1 


2187 


199 


28 


469 


2 


3352 


239 


36 


564 


3 


2678 


178 


ND 


447 


4 


1787 


113 


ND 


239 


5 


1886 


146 


ND 


348 


6 


2013 


167 


21 


457 


Mean ± SD 


2313 + 596 


174 + 43 


28 + 8 


421 ±112 



Table 3-40 : Concentrations in the rat plasma after i.v. administration of 
1 mg/kg of codeine. 



1 Sample 
number 


Codeine 
(ng/g) 


Morphine 
(ng/g) 


C6G 
(ng/g) 


M6G 
(ng/g) 


1 


247 


51 


27 


21 


2 


319 


49 


34 


25 


3 


276 


42 


23 


ND 


4 


231 


37 


21 


ND 


5 


198 


29 


ND 


ND 


6 


257 


45 


ND 


18 


Mean + SD 


255 + 41 


42 + 8 


26 + 6 


21 ±4 



Table 3-41 : Concentrations in the rat brain after i.v. administration 
of 1 mg/kg of codeine. 



Sample 
number 


C6G 
(ng/ml) 


1 


4736 


2 


3773 


3 


4062 


4 


5168 


5 


4183 


6 


3137 


Mean + SD 


4177 + 715 



Table 3-42 : Concentrations in the rat plasma after i.v. administration 
of 10 mg/kg codeine 6-glucuronide. 



120 



Sample 
number 


C6G 
(ng/g) 


M6G 

(ng/g) 


1 


197 


29 


2 


161 


ND 


3 


258 


27 


4 


396 


41 


5 


142 


ND 


6 


218 


35 


Mean ± SD 


229 ± 92 


33 ±6 



Table 3-43 : Concentrations in the rat brain after i.v. administration 
of 10 mg/kg codeine 6-glucuronide. 



Drug Administration 


Plasma 


Brain 


Codeine - ICV 


- 


CM 


C6G - ICV 


- 


C6G, M6G 


Codeine - SQ 


C, M, M3G 


C, M, C6G, M6G 


C6G - SQ 


- 


- 


Codeine - IV 


C, M, C6G, M3G 


C, M, C6G, M6G 


C6G - IV 


C6G 


C6G, M6G II 



Table 3-44 : Qualitative summary of results after administration of 
codeine and codeine 6-glucuronide by various routes. 



.■'*> ■y 



CHAPTER 4 
DISCUSSION 



The overall aim of this research project was to examine and compare the 
analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide. 
To achieve this objective the following hypotheses were formulated. 

1. Codeine 6-glucuronide, like morphine 6-glucuronide, possesses 
analgesic activity. 

2. The glucuronide metabolites of codeine and morphine are less 
immunosuppressive than their parent compounds. 

The rat was found to be a good model for the analgesia studies since 
negligible amounts of codeine 6-glucuronide are present in the rat plasma and 
urine after codeine administration. This allowed direct comparison of the 
antinociceptive activities of codeine 6-glucuronide with codeine without the 
contribution of codeine 6-glucuronide to the analgesia of codeine. 

An HPLC-UV based method was successfully developed for the analysis 
of codeine, morphine, codeine 6-glucuronide, morphine 6-glucuronide and 
morphine 3- glucuronide in biological samples, that is, human urine, rat plasma 
and rat brain. An efficient solid phase extraction method was used to obtain 
clean extracts and excellent recoveries in excess of 80% for all the compounds 
of interest. Calibration curves for each drug were linear over the concentration 
range 0-500 ng/ml in all the biological samples with a correlation coefficient of 
0.99 or better. 



121 



122 



During the synthetic procedure, an intermediate compound formed after 
the first reaction step was isolated and characterized. This intermediate 
represents the glucuronic acid moiety attached to the 6-position of codeine with 
intact acetyl/methyl groups. It appears to be comparatively more lipophilic than 
codeine 6-glucuronide and should be capable of crossing the BBB more easily. 
In these studies the exhibited antinociceptive activity which was similar to that of 
codeine 6-glucuronide itself. This indicates that this kind of "prodrug" might be a 
viable approach for delivering the potent glucuronides across the BBB. 

Intracerebroventricular administrations of morphine, codeine, codeine 6- 
glucuronide and the intermediate resulted in significant analgesic responses. All 
the compounds exhibited greater areas under the effect curve (AUECs) 
compared to saline treatment (p<0.01). Morphine (dose = 5 ^g) AUEC value was 
seen to be statistically higher than codeine 6-glucuronide (dose = 10 ^g), 
intermediate (dose = 10 |ag) and codeine (dose = 100 ^g), with p< 0.01. Both 
codeine 6-glucuronide and the intermediate exhibited larger AUECs compared to 
codeine (p<0.05). However, there were no statistically significant differences 
between codeine 6-glucuronide and the intermediate. Using this method, the 
rank order of effectiveness based on the AUEC values was morphine > codeine 
6-glucuronide z intermediate > codeine > saline. 

The analgesic effects of codeine 6-glucuronide and the intermediate were 
not statistically different from saline treatment after the subcutaneous studies. 
This lack of activity is probably associated with the limited ability of the polar 
glucuronides to cross the BBB and the poor absorption of these hydrophilic 
molecules from the subcutaneous tissue into the systemic circulation. Codeine 
(dose = 10 mg/kg) exhibited a statistically significant AUEC (p<0.01) compared 



123 



to codeine 6-glucuronide (dose = 10 mg/kg), intermediate (dose = 10 mg/kg) and 
saline treatments. There were no significant differences among the other 
treatments, that is, codeine 6-glucuronide, intermediate and saline. Comparison 
of total AUEC values indicated that at the dose administered, the order of 
effectiveness was codeine » codeine 6 -glucuronide z intermediate r saline. 

The intravenous route was chosen in order to remove one of the rate- 
limiting factors, that is, absorption. There was a significant increase in the 
responses associated with both codeine 6-glucuronide (dose = 10 mg/kg) and 
the intermediate (dose = 10 mg/kg). Codeine (dose = 10 mg/kg) also produced a 
greater response by this route compared to the subcutaneous route. This 
indicates that absorption of these compounds from the subcutaneous tissue can 
represent a significant barrier to the pharmacodynamic responses. All of the 
compounds exhibited greater areas under the effect curve compared to saline 
treatment (p<0.01). The codeine AUEC value was higher than codeine 6- 
glucuronide and the intermediate (p<0.01). However, there were no significant 
differences between the AUECs of codeine 6-glucuronide and the intermediate. 
Comparison of total AUEC values indicate that at the dose administered, the 
order of effectiveness was codeine > codeine 6 -glucuronide ~ intermediate> 
saline. 

All four agents tested, that is, codeine, morphine, morphine 6-glucuronide 
and codeine 6-glucuronide, demonstrated an immunomodulatory effect in all of 
the three different in vitro systems. Cell viability was greater than 98% in all 
cases as determined by the tryphan blue staining method. The differences 
between the effects of codeine and its 6-glucuronide in the PHA assay were 
more apparent at lower concentrations, that is, 0.156 to 0.625 jig/ml (p<0.01). 



124 



This data suggests that both codeine and codeine 6-glucuronide inhibit the T 
cell responses via the first signal activation pathway, which is mediated through 
interleukin-2 and calcium channel pathways. 

In the case of PMA-stimulated lymphocytes, the 6-glucuronide showed no 
inhibition in the lower, physiologically relevant concentrations (0.156 and 0.313 
|ig/ml) compared to codeine. The 6-glucuronide demonstrated significantly less 
inhibition than codeine in all concentrations tested (p< 0.05). Codeine and 
codeine 6-glucuronide also inhibited the MLR-stimulated lymphocytes in a dose- 
dependent fashion. However, there was no statistical difference between the 
effects of the two agents. 

Morphine and its 6-glucuronide also inhibited the PHA-stimulated cells in 
a concentration-dependent manner. Morphine 6-glucuronide, like codeine 6- 
glucuronide, produced almost no inhibition in the lower concentration range. In 
the concentration range of 0.156-2.5 iig/ml, the inhibitory effects of the 
glucuronide metabolite were less compared to its parent compound (p<0.05). As 
in the case of codeine and codeine 6-glucuronide, the differences were more 
apparent in the lower concentrations, that is, 0.156 to 0.625 ^g/ml (p<0.01). 

In the case of PMA-stimulated lymphocytes, morphine and morphine 6- 
glucuronide demonstrated almost negligible inhibition at lower concentrations 
(<1.25 ^g/ml).. At higher concentrations, however, both compounds showed a 
marked inhibition of proliferation. These compounds also showed inhibition of the 
mixed lymphocyte reaction (MLR). The 6-glucuronide of morphine showed less 
immunosuppression compared to morphine over the entire concentration range 
(0.156-10 |ig/ml). The decreased immunosuppressive effects of morphine 6- 



125 



glucuronide in comparison to morphine were statistically significant at 
concentrations below 0.625 |ig/ml (p<0.05). 

Both morphine and codeine exert their effects by interacting with the ^ 
opioid receptors in the central nervous system. The stronger analgesic activity of 
morphine compared to codeine is usually attributed to its high receptor binding 
affinity. In this study, the affinity profiles of morphine, codeine, codeine 6- 
glucuronide and the intermediate towards the ^ opioid receptor were examined. 
It was seen that morphine exhibited the highest affinity, with an IC50 value of 1.8 
± 0.6. In the case of codeine, however, the binding curve was shifted significantly 
to the right of the concentration range (IC50 = 1.5 + 0.4 jaM), indicating a much 
weaker affinity for the n receptor. This is in agreement with reports in the 
literature, which suggest a 1000 to 3000 fold less affinity for codeine compared 
to morphine (Chen et al., 1990 ; Mignat et al., 1995). Codeine 6-glucuronide 
exhibited slightly lower binding compared to codeine (IC50 = 4.4 + 0.2 |iM). This 
indicated that glucuronidation of codeine did not change the receptor affinity 
significantly. The intermediate, on the other hand, exhibited a higher ^ receptor 
binding compared to codeine and codeine 6-glucuronide (IC50 = 0.13 ± 0.06 ^M). 
This enhanced binding might be due to the fact that the intermediate is more 
lipophilic in nature than codeine and codeine 6-glucuronide. 

After intracerebroventricular administration of codeine, some of the 
codeine was converted into morphine in the brain. No codeine 6-glucuronide or 
any morphine glucuronides were detected. There was no drug detected in the 
plasma. When codeine 6-glucuronide was administered, there was a partial 
formation of morphine 6-glucuronide from codeine 6-glucuronide. As in the case 
of codeine administration, no drug was detected in the plasma. 



126 



After subcutaneous administration of codeine, it was seen that some of 
the codeine was converted into morphine in the plasma. Morphine 3-glucuronlde 
was also detected in the plasma, but no morphine 6-glucuronide or codeine 6- 
glucuronide were detected. In the brain, however, both morphine 6-glucuronide 
and codeine 6-glucuronide were detected. This indicates that the glucuronides 
can cross from the blood to the brain. In the case of subcutaneous administration 
of codeine 6-glucuronide, however, no drugs could be detected either in the 
plasma or the brain. This is most likely due to the very poor absorption of 
codeine 6-glucuronide from the subcutaneous tissue. 

After intravenous administration of codeine, some codeine was converted 
into morphine. Morphine 3-glucuronide was also detected in the plasma, but not 
morphine 6-glucuronide. There was also a small amount of codeine 6- 
glucuronide detected in the plasma. In the brain, both morphine 6-glucuronide 
and codeine 6-glucuronide were present. Codeine 6-glucuronide administration 
resulted in the presence of only codeine 6-glucuronide in the plasma, showing 
that it is stable in the plasma and does not convert to either codeine, morphine or 
morphine 6-glucuronide. In the brain, some codeine 6-glucuronide was seen to 
be 0-demethylated to morphine 6-glucuronide. Therefore, the analgesic 
responses observed in the rats would appear to be associated with codeine 6- 
glucuronide and to some extent, morphine 6-glucuronide. 

Codeine, although a chemical congener of morphine, is more widely used 
as an analgesic since it has substantially less potential for the development of 
dependence. It has been suggested that the analgesic action of codeine is 
associated with its conversion to morphine in the body. If this was the case 
however, one would expect to see relatively high concentrations of morphine in 



127 



the plasma and urine after codeine administration. However, only small amounts 
of morphine were detected after oral administration of codeine by various 
investigators (Quiding et al., 1986 ; Shah and Mason, 1990 a ; Persson et al., 
1992). In addition, urinary excretion of morphine has been reported to be only 
0.2-0.8% of the total codeine dose (Yue et al, 1990 a, b ; Chen et al., 1991). This 
is consistent with the report of Persson et al. (1992) that the ratio of the AUG of 
morphine to codeine is only 3%. Sindrup et al. (1990) found morphine 
concentrations of up to 13 ng/ml in extensive metabolizers 90 minutes after oral 
administration of 75 mg of codeine. In contrast, Findlay et al. (1986) and Guay et 
al. (1987) have reported concentrations of morphine up to 16 ng/ml after an oral 
dose of 60 mg of codeine, which theoretically could contribute to the analgesic 
effect of codeine. 

Although these reports regarding morphine levels after codeine treatment 
are contradictory, it is important to recognize that high morphine concentrations 
were all obtained using radioimmunoassays for morphine (Findlay et al., 1986 ; 
Guay et al., 1987), while other studies in which low or undetectable 
concentrations were reported used either HPLC (Chen et al., 1991 ; Shah and 
Mason, 1991 ; Yue et al., 1990 a, b) or GC-MS (Quiding et al., 1986) techniques. 
The apparent high concentrations of morphine may therefore be associated with 
cross-reactivity of the antibody to other opiates. Thus, in view of the low 
morphine concentrations it is most likely that its contribution to the analgesic 
effect of codeine is small. However, it has been hypothesized that O- 
demethylation to morphine may take place in the vicinity of receptors in the CNS 
(Chen et al., 1990). It has also been suggested that codeine may act on different 



128 



opioid receptors (Neil, 1984) and, therefore, may itself mediate a large part of its 
analgesic effect. 

There has been no documented study regarding the activity of codeine 6- 
glucuronide, the major metabolite in some species including humans. In contrast 
to the low concentrations of morphine produced after codeine administration in 
humans, concentrations of codeine 6-glucuronide have been reported to be 10- 
1 5 times those of codeine itself. We have shown that not only does codeine 6- 
glucuronide cross the blood brain barrier, it also has potent analgesic activity. 
This is in agreement with previous work describing the activity of morphine 6- 
glucuronide. 

Yoshimura et al. (1973) reported that both morphine 6-glucuronide and 
morphine 3-glucuronide could cross the blood brain barrier and act on the opioid 
receptors in the brain of rats. Other animal studies done in both rat and mouse 
have shown that morphine 6-glucuronide has a higher affinity for p opioid 
receptors than morphine itself (Abbott and Palmour, 1988 ; Pasternak et al., 
1980 ; Paul et al., 1989 ; Gong et al., 1991) and is 2-4 times more potent as an 
analgesic than morphine when injected subcutaneously as assessed by 
antinociceptive tests, that is, tail flick and hot plate tests (Gong et al., 1992 ; 
Hannaetal., 1990). 

Morphine 3-glucuronide, on the other hand, was found to have a very low 
affinity for |j opioid receptors in the brain and did not exhibit any analgesic effect 
when administered in pain-induced rat and mouse. Direct clinical evidence for 
the analgesic activity of morphine 6-glucuronide has been reported in man 
(Osborne et al., 1988 , 1990). It was seen that lower doses of morphine 6- 
glucuronide were needed compared to morphine for the same extent of pain 



129 



relief in cancer patients (Hanna et a!., 1990 ; 1991). Our results show that like 
morphine 6-glucuronide, codeine 6-glucuronide possesses analgesic activity of 
its own. . 'y ■> ■ 

The concept of opiates as imnnunomodulatory agents has long been 
recognized. It is well known that cell-mediated immune function Is impaired in 
heroin and opiate addicts (Brown et al., 1974) and that the major cell types 
involved in cell-mediated immunity, that is, T lymphocytes and mononuclear 
phagocytes, possess opiate receptors (Wybran et al., 1979 ; McDonough et a!., 
1980 ; Lopker et al., 1980). Opiate addicts have been shown to exhibit an 
increased incidence of bacterial, protozoal, fungal and viral infections (Bryant et 
al., 1990). Until recently, this was attributed to the sharing of unsterilized and 
contaminated needles by the addicts. However, there is now a considerable 
body of evidence suggesting that opiates themselves cause suppression of 
various immunological endpoints. 

The immunosuppressive effects of opiates may have potentially important 
clinical relevance, particularly in the case of individuals having a high degree of 
susceptibility towards infectious diseases. Effects on immunocompetence is a 
major area of concern when dealing with organ transplants, burns, cancer, 
rheumatoid arthritis and other autoimmune diseases. 

The relationship between the receptor binding affinity of codeine 6- 
glucuronide and its in vivo potency seems to be inconsistent. The glucuronide is 
analgesically much more active than its parent compound, but its receptor affinity 
does not reflect this observation. There are a few possible explanations. First, 
binding studies performed with brain homogenates do not necessarily represent 
the true physiological conditions. At best they can give a good approximation 



130 



regarding the activity of the compounds. They only indicate the binding of 
compounds to receptors under the specified conditions of the assay. However, 
this does not accurately estimate the efficacy of the compounds in vivo in 
producing a physiological response. Second, differences in the distribution, 
metabolism and elimination of these compounds can play a major role in 
modulating the pharmacodynamic responses. This too cannot be taken into 
account in binding studies. Third, these compounds may possess higher affinity 
towards other opioid (delta, kappa etc.) or even non-opioid receptors and this 
interaction may account for the enhanced analgesic potency of codeine 6- 
glucuronide in vivo. The lesser affinity of the glucuronide for the ^ receptor may 
also account for its lower immunosuppressive effects. 

Shashoua et al. (1986) have identified UDP-glucuronlc acid, a co- 
substrate for the glucuronidation reaction, in the rodent brain. There is also 
evidence of the presence of glucuronosyl transferase enzymes in the brain of 
rodents and humans (Wahlstrom et al., 1988). This raises the possibility that 
glucuronidation occurs in the brain. At present this seems unlikely and further 
investigation needs to be done in order to validate this hypothesis. It is more 
reasonable to assume that codeine 6-glucuronide produced in the liver is able to 
cross the blood brain barrier by an, as yet, unknown mechanism. 

The unexpected behavior of the hydrophilic conjugates of codeine and 
morphine with regards to crossing the blood brain barrier was partly explained by 
Carrupt et al. (1991). It was suggested that glucuronides existed in a 
conformational equilibrium between their hydrophilic and lipophilic forms, 
depending on the surrounding media. In the vicinity of the BBB the glucuronides 
were seen to be present in their lipophilic form which allowed easier penetration 



131 



into the brain. As the glucuronides circulating in the blood were in much higher 
concentration compared to their parent compounds, this caused a sufficient 
concentration gradient to be established, so as to allow the glucuronides to 
passively diffuse across the blood brain barrier (Barjavel et al., 1995). 

In conclusion, the data presented in this thesis suggests that the 
glucuronide metabolites of codeine and morphine may be more beneficial in the 
treatment of patients with low immunocompetence experiencing clinical pain 
during trauma and even after major surgery. Hence, both codeine 6-glucuronide 
and morphine 6-glucuronide, along with related compounds like the intermediate, 
deserve further assessment as potential agents for clinical pain management in 
the case of patients with a compromised immune function. 



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BIOGRAPHICAL SKETCH 

Vinayak Jaya Srinivasan was born on the 24th of April, 1969, in Kolar, 
India. He obtained his bachelors degree in pharmacy from the Institute of 
Technology - Banaras Hindu University in May, 1991. He started graduate 
studies in the College of Pharmacy at the University of Florida in August, 1991. 
After graduation, he plans to pursue a career in pharmaceutical research. 



' : , i ^- '. " i ' ' ' ^ '. 



ij 






149 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor of Philosophy. 



"^ ^T^.v5^ 



Ian R. Tebbett, Chair 

Associate Professor of Pharmaceutics 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor of Philosophy. 

Donna Wielbo 

Assistant Professor of Pharmaceutics 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor of Philosophy. 



U 01)^^4- 



Hartmut Derendorf 
Professor of Pharmaceutics 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor of Philososhy. 




Kenneth Sloan 

Professor of Medicinal Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentatkm^nd Is fully adeqtratevNin scope 
and quality, as a dissertation for the degree of Doctor of Ph]losophy^ 



(j><~<K.M^ I - ' ^4-/l..-V--c4-t>v--X 



Roger Bertholf 

Associate Professor of Pathology 
and Laboratory Medicine 



This dissertation was submitted to the Graduate Faculty of the College of 
Pharmacy and to the Graduate School and was accepted as partial fulfillment of 
the requirements for the degree of Doctor of Philosophy. / 



May, 1996 



Dean, College of Pharmacy 



Dean, Graduate School 




UNIVERSITY OF FLORIDA 



3 1262 08555 3195